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

journal of functional foods 14 (2015) 692–701

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Characterisation of anthocyanins and proanthocyanidins of adzuki bean extracts and their antioxidant activity Kyu-Ho Han a, Tomoko Kitano-Okada a,b, Jeong-Min Seo c, Sun-Ju Kim c, Keiko Sasaki d, Ken-ichiro Shimada a, Michihiro Fukushima a,* a

Department of Food Science, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan b Cosmo Foods Co., Ltd, Tokyo 103-0001, Japan c Department of Bio-Environmental Chemistry, Chungnam National University, Daehak-ro 99, Yusung-gu, Daejon 305-764, Republic of Korea d Hokkaido Tokachi Area Regional Food Processing Technology Center, Obihiro, Hokkaido 080-2462, Japan

A R T I C L E

I N F O

A B S T R A C T

Article history:

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

Received 1 September 2014

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

Received in revised form 6 February

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

2015

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

Accepted 9 February 2015

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

Available online

associated with polyphenol polymerization. Anthocyanins also contributed to the pigmentation of the purified adzuki bean extracts. Especially, two anthocyanin compounds, peonidin-

Keywords:

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

Vigna angularis

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

Anthocyanins

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

Proanthocyanidins

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

Pigments

dants and potential dyes.

Antioxidants

1.

Introduction

Legumes are a staple food in many countries and an excellent source of natural bioactive compounds. Among them, red adzuki bean (Vigna angularis) is cultivated throughout East Asia, where it is traditionally used for making bean pastes for use in confectioneries. As high polyphenol content in kidney bean (Phaseolus vulgaris L.) seed coats have been observed (Chen et al.,

© 2015 Elsevier Ltd. All rights reserved.

2014), the adzuki bean seed coat is also high in polyphenols (Lin & Lai, 2006); these polyphenols have been dominantly identified to contain catechin glycosides, quercetin glycosides, myricetin 3-rhamnoside, anthocyanin, and procyanidin dimers (Amarowicz, Estrella, Hernandez, & Troszynaska, 2008; Ariga, Koshiyama, & Fukushima, 1988). The bioactive compounds in the adzuki bean seed coat have received significant interest because of their health-promoting antioxidant properties (Lin & 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 0808555, Japan. Tel.: +81 155 49 5557; fax: +81 155 49 5577. E-mail address: [email protected] (M. Fukushima). Chemical compounds: Peonidin-3-rutinoside (CID 44256842); Pelargonidin-3-O-glucoside (CID 443648); Malvidin-3-O-glucoside (CID 443652); (+)-catechin (CID 9064). http://dx.doi.org/10.1016/j.jff.2015.02.018 1756-4646/© 2015 Elsevier Ltd. All rights reserved.

journal of functional foods 14 (2015) 692–701

reported to inhibit the formation of malonaldehyde (Lee, Mitchell, & Shibamoto, 2000), which is a maker for oxidative stress. A decrease in vascular oxidative stress and inflammation has also been reported in rats fed with the polyphenolcontaining adzuki bean seed coat (Mukai & Sato, 2011). Furthermore, Kitano-Okada et al. (2012) have shown in vitro that extracts from adzuki bean seed coat inhibit the activity of pancreatic lipase. These inhibitions would explain the results of a clinical trial that suggests that the consumption of the adzuki bean is linked to a reduced risk of lifestyle-related diseases in humans (Maruyama et al., 2008). To manufacture the adzuki bean paste, the beans are boiled in water, which is then generally discarded after completing the boiling process. However, the pigments contained in this water are receiving increased interest because of the need to maximize the utilization of natural resources in order to decrease the associated carbon footprint. Moreover, the food industry is experiencing an increasing demand for natural alternatives to replace synthetic food additives (e.g., antioxidants) (Amarowicz, Naczk, & Shahidi, 2000). In addition, adzuki bean extract is a potential source of pigments (food colourants) because the water used to boil the beans becomes strongly red or purple. Therefore, a novel extract powder purified from the water of adzuki bean paste production was developed and introduced in Japan to better utilize the resource (Adzuki-nomoto, Cosmo Foods Co., Ltd, Tokyo, Japan). The extract powder is highly purified and rich in natural polyphenols (Kitano-Okada et al., 2012). Interestingly, hot air-exposed purified adzuki bean extract rather maintains consistent colour strength than the normal purified adzuki bean extract because polyphenols in the adzuki bean extract were polymerized by oxidation, and both the extracts are stable to light, heat, and pH changes. However, information on responsible compounds for pigmentation as well as the potent antioxidative activity of the different purified adzuki bean extract powders is very limited. Therefore, the objective of this investigation was to compare the different purified adzuki bean extract powders from industrial residue in order to reveal their bioactive compounds for pigmentation and in vitro functional attributes such as reducing power and radical scavenging activity assay. For this, the antioxidant activity was compared with (+)-catechin as standard polyphenol.

2.

Materials and methods

2.1.

Reagents, chemicals, and standards

Calibrations were performed by using standard compounds (catechin, cyanidin-3-O-glucoside, pelargonidin-3-O-glucoside, peonidin-3-O-glucoside, and malvidin-3-O-glucoside) from Sigma-Aldrich (St Louis, MO, USA). Solvents (ethanol, butanol, acetonitrile and formic acid) and concentrated hydrochloric acid (HCl) were purchased from Wako Chemical Co., Ltd. (Tokyo, Japan). Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), malondialdehyde, 2,2-azobis(2-methylpropionamide) dihydrochloride (AAPH), 2,2-diphenyl-1-picrylhydrazyl (DPPH), thiobarbituric acid, and Folin–Ciocalteu phenol reagent were obtained from Sigma-Aldrich. Ferric ammonium sulphate and ferrozine were purchased from Kishida Chemical Co., Ltd.

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(Osaka, Japan). All other chemicals were purchased from Kanto Chemical Co., Ltd. (Tokyo, Japan).

2.2.

Adzuki bean extract powder (AEP)

The adzuki bean extract powder (AEP; also known as Adzukino-moto) made from the simmering water obtained during the sweetened adzuki bean paste production was kindly supplied by Cosmo Foods. The extract powder was made by the following procedure: first the bean was boiled and cooled. The water was collected from supernatant, adjusted at pH 4.0, treated with 0.005% pectinase HL (Yakult Pharmaceutical Industry Co., Ltd, Tokyo, Japan) and passed through a 50 mesh sieve to separate undigested materials. It was further applied to an ultrafiltration device to remove polymeric components and adjusted at pH 8.5. Then two different types of adzuki bean extract powder were made depending on the process with and without oxidative polymerization of polyphenols. One was sterilized (115 °C for 90 min) and spray-dried (AEP-1). The other was exposed to the air under heat treatment (90 °C for 5 h) and spray-dried (AEP-2).

2.3.

Total polyphenols and proanthocyanidins analyses

The total concentration of polyphenols in the AEP samples was determined according to the Folin–Ciocalteu’s method using (+)-catechin as a standard (Singleton, Orthofer, & Lamuela-Raventos, 1999). The absorbance was read at 750 nm using a spectrophotometer (1600-UV; Shimadzu, Kyoto, Japan). The results were expressed in mg of (+)-catechin equivalents to per gram of bean extract powder. All tests were performed in triplicate. For assay of total proanthocyanidins concentration in the AEP samples, the acid butanol method was employed (Porter, Hrstich, & Chan, 1986). In brief, 6.0 mL of the butanol–HCl reagent (95:5, v/v) were added to 1.0 mL of suitably diluted extract (1 mg/mL) in a screw cap test tube. Then 0.2 mL of ferric reagent (2% ferric ammonium sulphate in 2M HCl) was added to the solution. The container was capped, mixed and boiled for 30 min in a water bath. Similarly, a standard curve was prepared using procyanidin B-2 (Sigma-Aldrich) ranging from 0 to 0.25 mg/mL. After cooling, the absorbance was read at 550 nm using a spectrophotometer. The results were expressed in mg of procyanidin B-2 equivalents to per gram of bean extract powder. For quantification of oligomeric proanthocyanidins, the AEP sample was dissolved in water (1 mg/mL) and filtered using a 0.45 µm polytetrafluoroethylene (PTFE) hydrophilic syringe filter (Advantec Dismic-13HP, Toyo Roshi Co., Ltd., Tokyo, Japan). The filtrate was loaded into a Shimadzu Prominence HPLC system equipped with an LC-20AD pump (Shimadzu). The analytes were separated using a TSKgel ODS-80Ts column (4.6 mm × 250 mm, Tosoh, Tokyo, Japan) with photodiode array detector (SPDM20A, Shimadzu). Absorbance was monitored at 210–600 nm. The column oven temperature was set at 40 °C. The injection volume was 10 µL and the flow rate was 1.0 mL/min. The mobile phases were made of 0.05% trifluoroacetic acid (TFA) in water (v/v, mobile phase A) and 0.05% TFA in 90% acetonitrile (v/v, mobile phase B). Elution was used as a linear gradient from 5 to 35% B in 13 min, from 35 to 70% B in 20 min, and then 70%

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B in 25 min. Proanthocyanidin oligomers were quantified by comparing the HPLC peak area with that of flavangenol (Toyo Shinyaku Co., Ltd., Saga, Japan). The molecular weight (Mw) of AEP was determined by gel size exclusion chromatography. The filtrate (10 µL) was loaded into a Shimadzu gel permeation chromatography (GPC) system equipped with an LC-20AD pump and a refractive index detector (RID-10A, Shimadzu). The analytical double columns (7.6 mm × 250 mm, Shodex OHpak SB-806MHQ and OHpak SB802.5HQ, Tokyo, Japan) equipped with a security guard column (Shodex OHpak SB-G, 6.0 mm × 50 mm) were eluted with 20 mM lithium bromide in dimethylformamide at a flow rate of 0.6 mL/ min under the maximum operating pressure of 4.2 MPa. The column oven temperature was set at 40 °C. The standard curve of Mw was obtained using the following materials: catechin (Mw 290), procyanidin B-2 (Mw 578), and polystyrenes (Mws 780– 20000, Shodex SL 105, Tokyo, Japan). The average numberaverage molecular weight (Mn) and number-average degree of polymerization (DP) of the oligomers from the AEP sample was determined using GPC Software (LCsolution GPC Ver. 1.21, Shimadzu).

2.4.

Anthocyanin monomer analysis

The anthocyanins present in each sample were extracted and analyzed according to Chun et al. (2013). In brief, 5% formic acid in water (v/v, 2 mL) was added to sample tubes containing AEP (100 mg). The solution in each tube was vigorously mixed for 5 min, sonicated for 20 min, and centrifuged at 9200 × g for 15 min at 4 °C. The supernatant was filtered using a 0.45 µm PTFE hydrophilic syringe filter (Advantec Dismic-13HP). The anthocyanin containing filtrate was analyzed with an Agilent Technologies 1200 series HPLC (Palo Alto, CA, USA). The analytes were separated using a Synergi™ 4 µm Polar-RP 80A column (4.6 mm × 250 mm, Phenomenex, Torrance, CA, USA) equipped with a Security Guard Cartridges Kit AQ C18 column (3.0 mm × 4.0 mm, Phenomenex). The UV detector was set at 310 nm and the column oven temperature was set at 30 °C. The injection volume was 10 µL and the flow rate was 1.0 mL/ min. The mobile phases were made of water/formic acid (95:5, v/v, mobile phase A) and acetonitrile/formic acid (95:5, v/v, mobile phase B). A linear gradient programme was used as follows: A:B (95:5, v/v) to A:B (70:30, v/v) after 20 min, and then A:B (95:5) for an additional 10 min (modified from Wu & Prior, 2005). The anthocyanin components present in the AEP samples were identified by comparing the retention time (RT) of the authentic standards of the compounds (cyanidin-3-O-glucoside, pelargonidin-3-O-glucoside, peonidin-3-O-glucoside, and malvidin-3-O-glucoside). Some compounds were also identified by liquid chromatography–tandem mass spectrometry (LC– MS/MS) with a 4000 Qtrap LC–MS/MS system (Applied Biosystems, Foster City, CA, USA) using electrospray ionization (ESI) operating in the positive ion mode [M+H] +. The ESIMS was performed at a capillary temperature of 550 °C, an ion spray voltage of 5.5 kV, a curtain gas pressure of 20 psi and a spectra range (m/z) 100–1300 (scan time, 4.8 s). Nitrogen was used as a nebulizer gas with a pressure of 50 psi. The identified anthocyanin components present in the AEP samples were quantified by comparing the HPLC peak area with that of the authentic standards. Unidentified anthocyanin components

were quantified by comparing the HPLC peak area with that of the pelargonidin-3-O-glucoside, because such anthocyanidin is known to be a main component in adzuki bean (Wu & Prior, 2005).

2.5.

Total antioxidant activity

For stock solutions, an equivalent amount of polyphenols (6 mg of (+)-catechin) to that contained in the AEP samples was dissolved in distilled water (10 mL). The total antioxidant activity (TAA) was measured using a Randox kit (Randox Laboratories Ltd., Antrim, UK) according to the manufacturer’s instructions. The results are expressed as millimole Trolox per gram of polyphenols.

2.6.

Hydroxyl radical scavenging activity

The hydroxyl radical scavenging activity of the samples was determined according to 2-deoxyribose oxidation method described by Chung, Osawa, and Kawashiki (1997). All solutions except FeSO4–ethylenediaminetetraacetic acid (EDTA) were dissolved in 0.1 M sodium phosphate buffer (pH 7.4). In brief, the stock solution (100 µL, containing 600 µg/mL of polyphenols) was added to a freshly prepared mixture of 10 mM 2-deoxyribose, 10 mM FeSO4, and 10 mM EDTA (1:1:1, v/v/v) (600 µL). A sodium phosphate buffer (1 mL) and 10 mM H2O2 (200 µL) were added to the previous solution and put into a water bath at 37 °C. Exactly 4 h after incubation, 2.8% trichloroacetic acid (1.33 mL, v/v) and 1.0% thiobarbituric acid (670 µL, w/v) were added to the solution. The mixtures were then incubated at 80 °C for 10 min. After cooling, the absorbance (A) of the solution was measured at 532 nm using a spectrophotometer. The pentose 2-deoxyribose is oxidized by hydroxyl radical that is generated by Fe 2+ –EDTA to yield TBA/ malondialdehyde adduct (Chung et al., 1997). The hydroxyl radical scavenging activity of the AEP samples was calculated using the following formula: Inhibition (%) = [1 − (A sample − A sample blank )/(A control − Acontrol blank)] × 100, where Asample is the absorption of the sample solution and Acontrol is the absorption of the control solution. Both Ablanks are for the blank solutions not containing H2O2.

2.7.

Peroxyl radical scavenging activity

The peroxyl radical scavenging activity of the samples was measured by using the method described by López-Alarcón and Lissi (2005). In brief, the stock solution (100 µL, containing 600 µg/ mL of polyphenols) was mixed with 0.6 M AAPH (15 µL) and 60 mM pyrogallol red in the PBS buffer (pH 7.0) containing 30% ethanol (1 mL). The resulting solution was incubated in a water bath at 37 °C for 2 h, and the absorbance was then measured at 540 nm using a spectrophotometer. The peroxyl radical scavenging activity of the AEP samples was then calculated using the following formula: Inhibition (%) = [1 − (A sample − A sample blank )/(A control − Acontrol blank)] × 100, where Asample is the absorption of the sample solution and Acontrol is the absorption of the control solution (not containing the sample). Both Ablanks are for the blank solutions not containing AAPH.

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2.8.

DPPH radical scavenging activity

A diluted stock solution (300 µL, containing 0–108 µg/mL of polyphenols) was mixed with 0.4 mM DPPH (300 µL) and 0.2 M MES buffer (pH 6.0) containing 20% ethanol (600 µL). The standard solution used was 0.2 mM Trolox. The absorbance of the solution was measured at 520 nm and the radical scavenging activity was calculated using the following formula (Brand-Williams, Cuvelier, & Berset, 1995): DPPH radical scavenging activity (%) = (1 − Asample/Acontrol) × 100, where Asample is the absorption of the sample solution and Acontrol is the absorption of the control solution (not containing the sample).

2.9.

2.11.

Statistical analysis

The results reported here are the means of at least three measurements with standard error (SE). The data were analyzed using the general linear model procedure and the significance of differences was determined by the Tukey’s multiplerange test (SPSS 17 version, SPSS Institute, Armonk, NY, USA). A P-value of less than 0.01 was considered statistically significant.

Reducing power

The reducing capacity of the samples was measured using the method described by Oyaizu (1986). Briefly, a diluted stock solution (2.5 mL, containing 0–150 µg/mL of total polyphenols) was added to a freshly prepared solution of 0.2 M PBS buffer (pH 6.6) and 1% potassium ferricyanide (1:1, w/v). The mixture was incubated at 50 °C for 20 min, and then 10% trichloroacetic acid (2.5 mL, v/v) was added. The resulting mixture was centrifuged at 650 × g for 10 min. The upper layer of the solution (2.5 mL) was separated and mixed with distilled water (2.5 mL) and 0.1% FeCl3 (0.5 mL, w/v). The absorbance was read at 520 nm using a spectrophotometer.

2.10.

Chelating activity (%) = [1 − (Asample − Asample blank)/Acontrol] × 100, where Asample is the absorption of the sample solution, and Acontrol is the absorption of the control solution (not containing the sample). Asample blank is for background subtraction.

Chelating ability of ferrous ions

The chelating ability of ferrous ions in the samples was determined by using the method of Dinis, Madeira, and Almeida (1994). A diluted stock solution (740 µL, containing 0–600 µg/ mL of polyphenols) was mixed with 2 mM FeCl2 (20 µL). The reaction was initiated by adding 5 mM ferrozine (40 µL) and the mixture was shaken vigorously. After exactly 10 min, the absorbance of the solution was measured at 562 nm using a spectrophotometer. The blank sample was prepared without ferrozine for background subtraction.

3.

Results and discussion

3.1.

Total polyphenols and proanthocyanidins

The polyphenols in Vigna species of legume have been mainly identified as catechin, quercetin, myricetin, anthocyanin, and procyanidin dimers (Amarowicz et al., 2008). However, the polyphenols content in organic solvent extracts of adzuki bean varies. Sreerama, Takahashi, and Yamaki (2012) reported that the total polyphenols content in the methanol extract obtained from whole adzuki bean ranges from 35 to 73 mg of per gram dry weight equivalent of gallic acid. Furthermore, Amarowicz et al. (2008) reported that the total polyphenols concentration in the acetone extract of adzuki beans was approximately 9.0% (w/w). In this study, the total polyphenols content (equivalent to (+)-catechin) in samples AEP-1 and AEP-2 from the boiled water of adzuki bean paste production were 281 and 108 mg/g dry weight, respectively (Table 1), which suggests that the extract powders were highly purified and rich in polyphenols. Proanthocyanidins, which are oligomers or polymers of polyhydroxyflavan-3-ol units, are largely found in plant kingdom and food materials as pigment (Hosseinian & Mazza, 2009; Lee,

Table 1 – Total polyphenols, anthocyanins and proanthocyanidins contents in the purified extract of adzuki bean seed coat (n = 3). Peaka

Trivial names (anthocyanin)

1 Unknown 2 Unknown 3 Unknown 4 Unknown 5 Peonidin-3-rutinoside 6 Pelargonidin-3-O-glucoside 7 Malvidin-3-O-glucoside 8 Rutin or peonidin-3-(p-coumaroyl)glucoside Anthocyanins (mg/g, dry) Proanthocyanidins (mg proanthocyanidin B2 equivalent /g, dry) OPC (mg flavangenol equivalent/g, dry) Total phenolics (mg catechin equivalents/g, dry) a

RT (min)

6.7 8.3 10.0 10.3 10.9 13.7 15.0 15.9

AEP-1

AEP-2

mg/g, dry

mg/g, dry

b

2.61 6.20 4.55 5.64 43.8 7.90 5.89 20.7 97 157 – 281

2.76 1.93 – – 17.4 1.65 4.07 – 28 – 69 109

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

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journal of functional foods 14 (2015) 692–701

uV

4 Peak 4 (Monomer Mn, 274)

25000

20000

Standard

15000

1

10000

2 3

Peaks 1-3 (OPC Mn, 2290)

MW

RT (min)

Polystyrene 1

20000

23.027

Polystyrene 2

7350

23.605

Polystyrene 3

2340

25.332

Polystyrene 4

780

27.003

Procyanidin B-2

578

27.594

Catechin

290

28.389

5000

0

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

min

Fig. 1 – GPC profiles of monomer (peak 4) and oligomers (peaks 1–3) from the purified extract of adzuki bean powder-2. Peak 1 RT, 23.579; peak 2 RT, 25.208; peak 3 RT, 26.858; and peak 4 RT 27.742. RT, retention time (min). Mn, the average numberaverage molecular weight. OPC, oligomeric proanthocyanidin complexes.

2013). It was reported that some proanthocyanidins as dimeric procyanidins (B-type) are responsible for pigments of adzuki bean (Amarowicz et al., 2008; Ariga et al., 1988; Sreerama et al., 2012). In this study the total proanthocyanidins content (equivalent to procyanidin B-2) in the AEP-1 measured by the acid butanol assay was 157 mg/g dry weight (Table 1). However, we could not determine the total proanthocyanidins in the AEP-2 by this assay because the colour of reagents containing AEP-2 sample after heating was lower than the colour before heating. Although this assay is largely used for determining condensed tannins in foods (Porter et al., 1986), it is limited to quantify some interflavan bonded or doubly linked procyanidin oligomers (A-type) owing to them not being hydrolyzed upon heating (Hemingway, 1989; Watterson & Butler, 1983). Thus, it was hypothesized that the procyanidin dimers in the purified adzuki bean extract was more condensed and stable against acid and heat, and simultaneously became strongly reddish when an oxidative polymerization process was employed to make the AEP-2 sample. As expected, the polyphenols in the AEP-2 sample were polymerized with average DP 7.9 (excluding monomeric polyphenols), and the oligomeric proanthocyanidin complexes (equivalent to flavagenol) was 69 mg/g dry weight (Fig. 1). Therefore, the different polyphenols contents (or ratio of monomer to oligomer) in both the AEP samples might be explained by the polyphenols compounds being polymerized when a normal purified adzuki bean extract was exposed to the air under heat treatment.

3.2.

Anthocyanins

Anthocyanins are also responsible for the pigment of several plants. However, only trace amounts of anthocyanins have been identified as representative pigments in the adzuki bean seed coat. The anthocyanin in the adzuki bean was first identified

by Yoshida et al. (1996) using HPLC as cyanidin-3-O-(β-Dglucopyranosyl)-5-O-(β-glucopyranosyl) with a concentration of less than 0.1 mg/g of dried powder. Wu and Prior (2005) identified three anthocyanins such as cyanidin-3-glucoside, pelargonidin-3-glucoside, and pelargonidin-3-sambubioside at low concentrations (6.7 mg per 100 g of fresh weight) (Wu et al., 2006). Sreerama et al. (2012) reported that anthocyanin contents (mg cyanidin-3-glucoside equivalents per g of defatted flour) in the seed coat of adzuki bean are in the range 3.14– 7.94 mg/g dry weight. In contrast, our findings revealed that total anthocyanins in the purified adzuki bean extracts were considerably higher than those found in others. The total anthocyanins content in samples AEP-1 and AEP-2 were 97.3 and 27.8 mg/g dry weight, respectively (Table 1). HPLC chromatogram showed that the adzuki bean extract powder contains eight (Fig. 2A) and five peaks (Fig. 2B) for the AEP-1 and AEP-2 samples, respectively. The anthocyanin peaks were eluted at 6.7, 8.3, 10.0, 10.3, 10.9, 13.7, 15.0, and 15.9 min, respectively (Table 1). The authentic standard of cyanidin-3-glucoside had a RT of 13.2 min; however, cyanidin-3-glucoside in the adzuki bean extract powder could not be identified when compared with an authentic standard (Fig. 2). Peaks one to four are those of unknown compounds while peak five was identified as peonidin-3-rutinoside ([M+H]+, m/z 577/179) by mass spectroscopy (Fig. 3A). Peaks six and seven were identified as pelargonidin-3-O-glucoside and malvidin-3-O-glucoside, respectively, by comparing with the RT of authentic standards. Identification of peak eight, not found in the AEP-2 sample, was complicated by its similar molecular weight between quercetin3-rutinside (rutin) and peonidin 3-(p-coumaroyl) glucoside, showing to have MS data ([M+H]+ m/z, 609/301) (Fig. 3B). To our knowledge, no published data are available for malvidin-3-Oglucoside and peonidin-3-rutinoside as pigments in the adzuki bean seed coat. Furthermore, these chromatograms clearly

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

demonstrate that the class of anthocyanin compounds contained in the AEP samples differs depending on the process with and without oxidative polymerization which was carried out by exposing extracts to the air under heat treatment.

3.3.

Total antioxidant activity (TAA)

Due to the difficulties in measuring the antioxidant capacity of individual compounds in a complex mixture, several estimations including the Trolox equivalent (TE) value have been broadly used to represent the antioxidant capacity of foods, beverages, and supplements. The TAA, which is expressed as TE value, of leguminous pigment extracts from whole bean powder ranged from 0.30 to 1.76 mmol Trolox equivalent to per

gram bean extract (Amarowicz, Troszynska, Barylko-Pikielna, & Shahidi, 2005). Especially, a higher TAA has been reported for the extracts of polyphenols from adzuki bean, which TE value is higher than those obtained from others (Amarowicz et al., 2005). The TE values in this study were extrapolated to those determined by Amarowicz et al. (2005), with AEP-1 and AEP-2 samples having approximately 0.908 and 0.359 mmol Trolox/g of powder, respectively. The different TAA between AEP-1 and AEP-2 samples was owing to the presence of different concentrations of polyphenols in the AEP samples. Although the total amount of total polyphenols was higher in the AEP-1 sample than the AEP-2 sample (Table 1), a higher TAA was measured for AEP-2 than AEP-1 when the TE value was based on the amounts of polyphenols in the AEP samples, 3.30

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

versus 3.23 mmol Trolox/g of polyphenol, respectively (Table 2). This difference strongly implies that the polyphenols present in the extract powder were not degraded by the oxidation process used for AEP-2, but rather condensed in their active forms. These results are in agreement with a recent study (Li et al., 2015) that found a higher TAA for polymerized polyphenols (average DPs of approximately 3–6) than dimer polyphenols.

3.4.

Radical scavenging activity

Peroxyl radical inhibition was determined using the AAPH method (López-Alarcón & Lissi, 2005). AAPH is a water-soluble azo compound that is used extensively as a free-radical generator in the characterization of antioxidants. As shown in the Table 2, peroxyl radical inhibition was significantly (P < 0.01)

higher in the order AEP-1 > AEP-2 > (+)-catechin. On the other hand, samples with the same equivalent concentration of polyphenols had a relatively lower hydroxyl-radical scavenging activity than peroxyl radical inhibition; no significant difference was observed between the trials (Table 2). In addition, the radial-scavenging activity of the extracts was examined using the free radical, DPPH. As shown in Fig. 4, the DPPH radical scavenging activity increased directly with the concentration of polyphenols (from 0 to 108 µg per assay). The DPPH radical scavenging activity at the highest concentration (108 µg) was significantly higher (P < 0.01) in the order AEP-2 > AEP-1 > (+)catechin. The DPPH radical assay revealed that both the AEP samples showed higher antioxidant capacities than (+)catechin, with AEP-2 showing the highest value. This finding is in accordance with the findings of previous studies that

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Table 2 – Total antioxidant activity (TAA), and hydroxyl and peroxyl radical scavenging activities of adzuki bean extracts powder.

TAA (mmol Trolox/g polyphenol1) Hydroxyl radical inhibition2 (%) Peroxyl radical inhibition2 (%)

(+)-Catechin

AEP-1

AEP-2

3.34 ± 0.01a 2.87 ± 0.71a 14.5 ± 0.3c

3.23 ± 0.01b 8.40 ± 1.77a 61.9 ± 0.5a

3.30 ± 0.01a 4.92 ± 0.49a 48.8 ± 0.4b

1

Polyphenol concentration was equivalent to (+)-catechin. Amount of extract was prepared at an equivalent catechin concentration as 0.6 mg/mL. AEP-1, normal adzuki bean extracts powder; and AEP-2, polymerized adzuki bean extracts powder. Values are mean ± standard error of 3 replicates. Means within the same rows bearing different superscripts are significantly different (P < 0.01) by analysis using Tukey test.

2

reported that the polymerized fraction of the adzuki bean extract exhibited a greater radical scavenging activity (DPPH radical) than the low-molecular-weight fraction of polyphenols (Amarowicz et al., 2008; Sulaiman, Ibrahim, Kassim, & Sheh-Hong, 2011). A possible explanation for these results, recently reported by several researchers (Kurisawa, Chung, Uyama, & Kobayashi, 2003; Sulaiman et al., 2011), is that condensed phenols such as proanthocyanin are better free radical inhibitors (primary antioxidants) than monomeric phenols such as (+)-catechin.

3.5.

Reducing capacity

The reducing capacity of the samples is based on the reduction of the Fe3+ ion, where antioxidants are the reducing agents; thus, the reducing capacity is associated with antioxidant activity (Benzie & Strain, 1999). Compounds that are capable of donating a single electron or hydrogen atom for reduction might reduce oxidized intermediates. In this assay, the presence of an antioxidant in the extracts reduced the Fe3+/ferricyanide complex to Fe2+ ion (Fig. 5A). The reducing capacity of a variety of plant extracts, including those of fruits, tea, and legumes,

DPPH radical scavenging activity (%)

60

a AEP-1

50

AEP-2

b

Catechin

c

*

40

has been extensively studied (Li et al., 2015; Lin & Lai, 2006; Xiao et al., 2014). In a study by Lin and Lai (2006), the reducing power of various legumes was shown to be dependent on the content of phenolic compounds. However, in our study, the polyphenols concentration of the bean extract was adjusted so that it was equivalent to (+)-catechin; therefore, no correlation was found. These results (Fig. 5A) show that the absorbance values were remarkably higher for AEP-2 than for AEP-1 samples and (+)-catechin, strongly suggesting that oligomeric proanthocyanidins in the AEP-2 sample were more efficient reductants than low-molecular-weight polyphenols in the AEP-1 sample. Polymerized polyphenols (e.g., condensed tannin) exhibit a stronger reducing capacity than that of lowmolecular-weight and/or monomeric phenols (Pulido, Bravo, & Saura-Calixto, 2000) and the results are in agreement with the findings of our study. Interestingly, a comparison of the reducing capacity and peroxyl radical inhibitory capacity reveals that these properties are not correlated to the state of polymerization. One possible explanation for this observation can be drawn from the study of Simic and Jovanovic (1994). Their study demonstrated that lower redox potentials correlated to higher antioxidant efficiency against free radicals such as peroxyl or hydroxyl radicals (Simic & Jovanovic, 1994). This finding may explain the opposite observation between the reducing capacity and the peroxyl radical inhibitory capacity in AEP-2 samples compared to that of AEP-1 samples. These results provide important insights into the properties of polyphenols depending on their polymerization state.

30

3.6.

*

20 10 0 0

30

60

90

120

150

180

Contents (µg/assay)

Fig. 4 – Scavenging effect of adzuki bean extract powder on the DPPH radical, as measured by changes in absorbance at 520 nm. The experiments were performed in triplicate. Means with different letters (a–c) at each concentration level are significantly different (P < 0.01) by analysis using Tukey test. *P < 0.01 versus AEP-1. AEP-1, normal adzuki bean extracts powder; AEP-2, polymerized adzuki bean extracts powder.

Ferrous ion chelating activity

Fig. 5B shows the Fe2+ ion chelating activity (%) of AEPs and (+)-catechin at six concentration levels (0–450 µg/assay), whereby the activity increased depending on the concentration for all samples investigated. At all concentrations tested, the ferrous ion chelating activity of the AEP samples was far superior to that of (+)-catechin. At a concentration of 100 µg/assay, both the AEP samples showed approximately 96% chelating activity; however, (+)-catechin showed only 1.6% chelating activity. Andjelkovic et al. (2006) showed that the ability of phenolic compounds to chelate ferrous ions is far lower than that of EDTA. In contrast, Sreerama et al. (2012) showed that the ability of the adzuki bean extract to chelate ferrous ions was comparable to that of EDTA. However, some researchers have argued that metal chelation plays a smaller role in the overall

journal of functional foods 14 (2015) 692–701

3.0

100 AEP-1

2.5

AEP-2

*

Catechin

2.0

*

1.5

1.0

* (A)

*

0.5

0.0

Ferrous ion chelating activity (%)

Reducing capacity (Absorbance at 700 nm)

700

80

* 60

AEP-1

AEP-2

Catechin

*

40

20

(B)

*

0 0

50

100

150

0

50

100

150

200

250

300

350

400

450

Contents (µg/assay)

Contents (µg/assay)

Fig. 5 – Reducing capacity (A) and ferrous ion chelating activity (B). The experiments were performed in triplicate. *P < 0.01 versus AEP-1. AEP-1, normal adzuki bean extracts powder; AEP-2, polymerized adzuki bean extracts powder.

antioxidant activities of some polyphenols such as (+)-catechin (Rice-Evans, Miller, & Paganga, 1996). Furthermore, some proteins (Saiga, Soichi, & Nishimura, 2003) and oligosaccharides (Wang et al., 2007) can chelate metal ions. Thus, we could not conclude that polyphenols compounds such as anthocyanins and proanthocyanidins in the AEP samples were solely responsible for metal chelation, because AEP samples are a complex mixture of food ingredients (Kitano-Okada et al., 2012). Nevertheless, the results of this study indicate that AEP itself may serve as a potential source of chelating agents.

Acknowledgments This work was supported by a grant from the programme Cooperation of Innovative Technology and Advanced Research in the Evolution Area (CITY AREA, Development Stage) of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Appendix: Supplementary material 4.

Conclusions

This study determined that proanthocyanidins as well as anthocyanins were responsible for the pigment properties of the adzuki bean extract powders. The new anthocyanins in the adzuki bean extract powder were identified as malvidin-3-Oglucoside and peonidin-3-rutinoside. One of the more significant findings of this study was that the antioxidant capacity of both the AEP samples was superior to that of (+)-catechin with regard to ferrous ion chelating activity, although it is not conclusive whether only polyphenols were responsible for metal chelation. The reducing capacity of the AEP-2 sample was higher than that of (+)-catechin or AEP-1 sample. Furthermore, our findings suggest that the antioxidant activities of polyphenols containing oligomeric proanthocyanidins in the AEP-2 were greater than that of AEP-1. Thus, this study shows that the adzuki bean extract powder, especially AEP-2, which is generally discarded, is a valuable resource for naturally derived food additives with significant antioxidant activity. Thus, adzuki bean extract powders are promising alternatives to synthetic antioxidants and dyes, which are currently used in the food industry.

Supplementary data to this article can be found online at doi:10.1016/j.jff.2015.02.018.

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