ANTIOXIDANT ACTIVITY OF EXTRACT OF ADZUKI BEAN AND ITS FRACTIONS

ANTIOXIDANT ACTIVITY OF EXTRACT OF ADZUKI BEAN ANDITS FRACTIONS

RYSZARD AMAROWICZ1,3, ISABEL ESTRELLA2, TERESA HERNÁNDEZ2 andAGNIESZKA TROSZYNSKA2

1Department of Food ScienceInstitute of Animal Reproduction and Food Research of Polish Academy of Sciences

ul. Tuwima 10, 10-474 Olsztyn, Poland

2Instituto de Fermentaciones Industriales (CSIC)Juan de la Cierva, Madrid, Spain

Submitted for Publication September 27, 2006Revised Received and Accepted October 1, 2007

ABSTRACT

Phenolic compounds were extracted from adzuki bean using 80% (v/v)aqueous acetone. Crude extract was applied onto a Sephadex LH-20 column.Fraction I, consisting of low-molecular-weight phenolics, was eluted fromthe column using ethanol. Fraction II, consisting of tannins, was obtainedusing water/acetone (1:1; v/v) as the mobile phase. Phenolic compoundspresent in the crude extract and its fractions showed antioxidant and radicalscavenging properties as revealed using a b-carotene-linoleate model system,the total antioxidant activity (TAA) method, the 2,2-diphenyl-1-picrylhydrazyl(DPPH) radical scavenging activity and reducing power evaluation. Resultsof these assays showed highest values when tannins (fraction II) were tested.For example, the TAA of the tannin fraction was 4.17 mmol Trolox/mg,whereas extract and fraction I showed 1.76 and 1.40 mmol Trolox/mg,respectively. The content of total phenolics in fraction II was the highest(189 mg/g). The content of tannins in this fraction determined using the van-illin method and expressed as absorbance units at 500 nm/1 g was 213. Therewere 29 compounds (hydroxycinnamics, procyanidins, gallates, flavonols,dihydroflavonols, dihydrochalcones) identified in the crude extract using ahigh-performance liquid chromatography with photodiode array and massspectrometry detectors (HPLC-PAD-MS) method. Catechin and epicatechinglucosides, procyanidin dimers, myricetin and protocatechuic acid were thedominant phenolics in the extract.

3 Corresponding author. TEL: 48-89-523-4627; FAX: 48-89-524-0124; EMAIL: [email protected]

Journal of Food Lipids 15 (2008) 119–136. All Rights Reserved.© 2008, The Author(s)Journal compilation © 2008, Blackwell Publishing

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PRACTICAL APPLICATIONS

Results showing the antioxidant activity of adzuki bean extract and itsfraction can be useful for producers of healthy foods. The tannin fractionseparated from adzuki can be applied for production of natural antioxidantpreparations. The results of chemical analysis are valuable for databases offood phenolic compounds.

INTRODUCTION

Many phytochemicals found in whole plant foods can help preservevascular health and diminish cancer risk; direct antioxidant activity maymediate much of their benefit (Halliwell et al. 1992; McCarty 2004). Naturalantioxidants are compounds that detoxify reactive oxygen species and preventtheir damage to cellular macromolecules and organelles through differentmechanisms (Shahidi 2000).

Phenolic compounds belonging to natural antioxidants are secondarymetabolites commonly found in both edible and nonedible parts of plants.Evaluation of antioxidant activity of phenolic compounds from leguminousseeds has been of interest in recent years. Antioxidant activity has beenreported for extracts of legumes such as pea; white, green, red and navy beans;beach pea; lentils; everlasting pea; broad bean; faba bean; lima bean; Jackbean; vetch; adzuki bean; and cowpea (Ariga et al. 1988; Ariga and Hamano1990; Onyeneho and Hettiarachchy 1991; Tsuda et al. 1993; Amarowicz et al.1996a,b, 2001, 2008a,b; Carbonaro et al. 1996; Hempel and Bohm 1996; Raabet al. 1996; Yoshiki et al. 1996; Amarowicz and Raab 1997; De Mejía et al.1999; Zielinski 2002; Amarowicz and Troszynska 2003, 2004; Betancur-Ancona et al. 2004; Madhujith et al. 2004a,b; Randhir and Shetty 2004;Randhir et al. 2004; Dueñas et al. 2005, 2006; Madhujith and Shahidi2005a,b; Alsalvar et al. 2006; López-Amorós et al. 2006; Zhou and Yu 2006;Berger et al. 2007; Rocha-Guzmán et al. 2007a,b). Antioxidative and antiradi-cal activities of leguminous extracts have been investigated through storagestudies (Onyeneho and Hettiarachchy 1991; Madhujith et al. 2004a), ab-carotene-linoleate model system (Ariga and Hamano 1990; Amarowiczet al. 1996a,b, 2004c; Amarowicz and Troszynska 2003; Karamac et al. 2004),an EPR spin trapping method (Yoshiki et al. 1996), enhanced chemilumines-cence and photoluminescence (Raab et al. 1996; Amarowicz and Raab 1997),scavenging of 2.2′-azobis (2,4-dimethylvaleronitrite) (AMVN) radical (Arigaand Hamano 1990), 2,2′-azobis (2-amidopropane) hydrochloride (ABAP)radical (Zielinski 2002), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (Ama-rowicz and Troszynska 2003; Zhou and Yu 2006), 2,2′-azinobis-3-

120 R. AMAROWICZ ET AL.

ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radical (Amarowicz andTroszynska 2003; Zhou and Yu 2006), superoxide anion radical (Troszynskaand Kubicka 2001; Zhou and Yu 2006), peroxyl radical (Zhou and Yu 2006),reducing power (Amarowicz and Troszynska 2003), low-density lipoprotein(LDL) cholesterol oxidation (Madhujith and Shahidi 2005a) and Fe2+ chelatincapacity (Madhujith and Shahidi 2005b; Zhou and Yu 2006).

In recent years, evidence has been accumulating about the high antioxi-dant potential of tannins (Muir 1996; Amarowicz et al. 2000a,b, 2004a,b;Dueñas et al. 2003a). The high impact of tannins of leguminous seed extractshas previously been reported (Amarowicz and Troszynska 2003; Amarowiczet al. 2004a, 2008a; Dueñas et al. 2006). The objectives of this research wereto investigate the antioxidant and antiradical activities of an acetone extract ofadzuki beans and its low-molecular-weight and tannin fractions. The occur-rence of phenolics in the crude extract by HPLC-PAD-MS was also studied.

MATERIALS AND METHODS

Materials

All solvents used were of analytical grade. Methanol, acetone, ethanol,acetonitrile, potassium ferricyanide and trichloroacetic acid were acquiredfrom the P.O.Ch. Company (Gliwice, Poland). Butylated hydroxyanisole,b-carotene, linoleic acid, vanillin, Folin–Ciocalteau’s reagent, polyoxyethyl-enesorbitan monopalmitate (Tween 40), Sephadex LH-20 and DPPH radicalwere obtained from Sigma (Poznan, Poland). Protocatechuic acid, protocat-echuic aldehyde, trans p-coumaric acid, (+)-catechin, (–) epicatechin, dihyd-roquercetin, quercetin, quercetin 3-O-glucoside, quercetin 3-O-galactoside,quercetin 3-O-rutinoside, myricetin 3-O-rhamnoside, kaempferol 3-O-rutinoside, tryptophan, 2′,4,4′,6′-tetrahydroxydihydrochalcone-2′-O-b-glucoside (phloridzin) were purchased from Extrasynthese (Genay Cedex,France). The tryptophol was from Aldrich (Munich, Germany).

Seeds of adzuki bean were purchased from a local health food store(Olsztyn, Poland; seeds were imported to Poland from Japan).

Extraction

Bean seeds were ground in a coffee mill and then defatted with hexanesin a Soxhlet apparatus for 6 h. Phenolic compounds were extracted from theraw material using 80% (v/v) acetone at a solid to solvent ratio of 1:10 (w/v),at 50C for 30 min (Amarowicz et al. 1995). Extraction was carried out indark-colored flasks using a shaking water bath (Elpan 357, Wroclaw, Poland).The extraction was repeated twice, supernatants combined, and acetone was

121ANTIOXIDANT ACTIVITY OF ADZUKI BEAN

evaporated under vacuum at 40C in a rotary evaporator (Unipan 359P,Wroclaw, Poland); the remaining water solution was removed and lyophilized.The prepared extract was stored at -20C until used.

Column Chromatography

Separation of crude extracts into low-molecular-weight phenolics andtannin fraction was carried out according to the method described by Strum-eyer and Malin (1975). A 2-g portion of the crude extract was suspended in20 mL of 95% (v/v) ethanol and was applied onto a chromatographic column(5 ¥ 40 cm) packed with Sephadex LH-20 and equilibrated with 95% (v/v)ethanol. Low-molecular-weight phenolic compounds (fraction I) were elutedfrom the column using 1 L of 95% (v/v) ethanol. To obtain tannins (fractionII), the column was washed with 500 mL of 50% (v/v) acetone. Organicsolvents were evaporated and water was removed by lyophilization.

Total Phenolics

The content of total phenolic compounds in extract and each fraction wasestimated using the Folin–Ciocalteau’s phenol reagent (Naczk and Shahidi1989). (+)-Catechin was used as a standard.

Content of Tannins

The content of tannins in the crude extract and its fractions was deter-mined using the modified vanillin method assay (Price et al. 1978); resultswere expressed as absorbance units at 500 nm/g extract (A500/g).

Total Antioxidant Activity (TAA)

The determination of the TAA was carried out using a Randox kit(Randox Laboratories Ltd., Crumlin, U.K.) according to the procedure pro-vided by the supplier; a concentration of 2 mg extracts/mL methanol was usedin the assay. Results were expressed as micromole Trolox per milligram.

Antioxidant Activity in an Emulsion System

The antioxidant activity of the acetone extract of adzuki bean and itsfractions was determined in an emulsion system using the method describedby Miller (1971). Methanolic solutions (0.2 mL) containing 2 mg of crudeextract or fraction I or 1 mg of fraction II were added to a series of tubescontaining 5 mL of a prepared emulsion of linoleate and b-carotene stabilizedwith Tween 40 (Sigma). Immediately after the addition of emulsion to each


tube, the zero-time absorbance at 470 nm was recorded. Samples were kept ina water bath at 50C, and their absorbance values were recorded over a 120-minperiod at 15-min intervals.

Reducing Power

The reducing power of phenolics was determined as described by Oyaizu(1986). The suspension of the extract and fractions I and II in 1 mL of distilledwater was mixed with 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mLof 1% (w/v) potassium ferricyanide. The mixture was incubated at 50C for20 min. Following this, 2.5 mL of 10% (w/v) trichloroacetic acid was added,and the mixture was then centrifuged at 1,750 ¥ g for 10 min. A 2.5 mL aliquotof the upper layer was mixed with 2.5 mL of distilled water and 0.5 mL of0.1% (w/v) FeCl3; the absorbance of the mixture was read at 700 nm.

Scavenging of DPPH Radical

The scavenging effect of phenolics from the crude adzuki bean extractand its two fractions was monitored as described by Yen and Chen (1995). A0.1-mL methanolic solution containing 0.5–2.5 mg of extract or fraction I and0.02–0.1 mg fraction II was mixed with 2 mL of water and then added to amethanolic solution of DPPH (1-mM 0.25 mL). The mixture was vortexed for1 min, then left to stand at room temperature for 20 min, and the absorbance ofthis solution was subsequently read at 517 nm.

High-performance Liquid Chromatography with Photodiode ArrayDetector (HPLC-PAD) Analysis

The lyophilized extract (150 mg) was dissolved in 2 mL of 80% (v/v)methanol and filtered through a 0.45-mm cellulose acetate filter (Millipore,Billerica, MA) before HPLC analysis. The samples were prepared in duplicate.

The chromatographic system was equipped with an autoinjector, a qua-ternary pump, a photodiode-array detector 2001 (Waters, Milford, MA) andNova-Pak C18 (300 ¥ 3.9; 4 mm) column (Waters). The analytical conditionswere those described by Dueñas et al. (2004). Two mobile phases wereemployed for elution: (A) water/acetic acid (98:2, v/v) and (B) water/acetonitrile/acetic acid (78:20:2, v/v/v). The gradient profile was 0–55 min,100–20% A; 55–70 min, 20–10% A; 70–80 min, 10–5% A; and 80–100 min,100% B. The flow rate was 1 mL/min from the beginning to 55 min and1.2 mL/min from this point to the end. The column was re-equilibratedbetween injections with 10 mL of acetonitrile and 25 mL of the initial mobilephase. Detection was performed by scanning from 210 to 400 nm with anacquisition speed of 1 s. A volume of 100 mL was injected. The sample wasanalyzed in duplicate.


High-performance Liquid Chromatography with Mass SpectrometryDetector (HPLC-MS) Analysis

Mass spectra were obtained using a Hewlett Packard 1100 MSD (PaloAlto, CA) chromatograph equipped with an API source, using an electronspray ionization (ESI) interface and the conditions reported by Dueñas et al.(2004). The solvent gradient and column used were the same as those forHPLC-PAD. ESI conditions were as follows: negative mode, nitrogen wasused as the nebulizing gas, 40 psi, drying gas, 10 L/min at 340C; voltage atcapillary entrance, 4,000 V; and variable fragmentation voltage, 100 V (m/z200–1,000), 250 V (m/z 1,000–2,500). Mass spectra were recorded from m/z100 to m/z 2,500.

Identification and Quantification of Phenolic Compounds

Chromatographic peaks were identified by comparing the retention times,UV and electron spray ionization mass spectrometry (ESI-MS) spectra tothose of standards (standards used are listed in Materials). Other compoundswith UV spectra similar to those of hydroxycinnamates, procyanidins, gallates,flavonols, dihydroflavonols and dihydrochalcones were identified as theirderivatives. Their chemical structures were confirmed by HPLC-MS (ESI).

Quantification was made using the external standard method according tothe maximum of absorption of each compound. The calibration curves weremade by injection of different volumes of the standards from a stock solutionover the concentration range observed for each compound. The calibrationcurves were calculated as linear regressions of peak area versus standardconcentration. The trans-p-coumaric acid derivative was quantified usingthe calibration curve of the corresponding free phenolic acid, procyanidinsusing the calibration curve of (+)-catechin. Dihydrochalcone was expressed asphloridzin, and dihydroflavonols as dihydroquercetin.

RESULTS AND DISCUSSION

The content of total phenolics in fraction I was almost two times lowerthan that in the crude extract and more than three times lower than in fractionII (Table 1). A similar relation between the content of total phenolics in a crudeextract and its low-molecular-weight phenolics and tannin fractions wasobserved in the case of pea (Amarowicz et al. 2003), red bean (Amarowicz andTroszynska 2004), vetch (Amarowicz et al. 2008a) and red lentil (Amarowiczet al. 2008b). For Sephadex LH-20 column chromatography, the first solventused was ethanol. Ethanol can elute some phenolics together with sugars fromthe column. Because sugars were dominant compounds of the crude extract,


the content of phenolics in fraction I was low. A high content of total phenolicsin the tannin fraction, separated from the crude extract using a SephadexLH-20 column, was described previously for extracts of leguminous seedssuch as beach pea, faba bean (Amarowicz et al. 2000b), pea (Amarowicz andTroszynska 2003), vetch (Amarowicz et al. 2008b), red lentil (Amarowiczet al. 2008b), canola hulls and evening primrose (Amarowicz et al. 2000b).

The content of tannins, expressed as absorbance value at 500 nm/g, infraction II (213), was about two times higher than that in the crude extract(136). For the low-molecular-weight phenolic fraction, the observed vanillin-positive reaction could be due to catechin and other flavan-3-ols, which canelute from the Sephadex LH-20 column with ethanol (Amarowicz and Shahidi1995). The presence of tannins in leguminous seeds has been reported byseveral authors (Ariga et al. 1988; Ariga and Hamano 1990; Chavan et al.1999; Amarowicz et al. 2000b; De Pascuale et al. 2000; Dueñas et al. 2002,2003a, 2004; Madhujith et al. 2004a,b).

The highest value of the TAA was noted for the tannin fraction:4.17 mmol Trolox/mg (Table 1). Much less active was the crude extract(1.76 mmol Trolox/mg) and fraction I (1.40 mmol Trolox/mg). For the metha-nolic extracts of wheat, barley, rye and oat, Zielinski and Kozłowska (2000)reported lower values of TAA (0.054–0.222 mmol Trolox/mg) than thoseobtained in this study. For extract of seed coats from pea, TAA was3.6 mmol Trolox/mg (Troszynska and Kubicka 2001). Vetch extract and itslow-molecular-weight phenolics and tannin fractions exhibited TAA values of0.79, 0.40 and 6.40 mmol Trolox/mg, respectively (Amarowicz et al. 2008a).

The acetone extract of phenolic compounds from adzuki bean and frac-tions I and II exhibited antioxidant activity in a b-carotene-linoleate modelsystem (Fig. 1). The effect of the extract on the coupled oxidation of linoleicacid and b-carotene was the highest, but the addition of the tannin fraction wasonly 1 mg. On the other hand, these fractions might be quickly oxidized andtherefore could not be effective as an antioxidant in the emulsion model. Usingthe same method, similar or lower antioxidant activities were found for

TABLE 1.TOTAL PHENOLICS, TANNIN CONTENT AND TOTAL ANTIOXIDANT ACTIVITY IN

ADZUKI BEAN CRUDE EXTRACT AND ITS FRACTIONS

Analyzed material Total phenolics (mg/g) Tannins (A500/g) Total antioxidant activity(mmol Trolox/mg)

Crude extract 90 � 2 136 � 3 1.76 � 0.05Fraction I 58 � 1 45 � 1 1.40 � 0.04Fraction II 189 � 4 213 � 4 4.17 � 0.13

A500/g, absorbance units at 500 nm/1 g extract.


extracts of leguminous seeds such as faba bean, broad bean, lentil, beach peaand everlasting bean (Amarowicz et al. 1996a,b; Chavan et al. 1999).

The strong scavenging effect of tannins separated from the adzuki beanextract was confirmed in a DPPH assay (Fig. 2). The tannin fraction exhibitedantiradical activity several times higher than that of the crude extract. Thelow-molecular-weight phenolic fraction was a weaker scavenger of the DPPHradical. The scavenging effect of condensed tannins from beach pea, canolahulls, evening primrose and faba bean on the DPPH radical was describedby Amarowicz et al. (2000b). The radical scavenging activity of condensedtannins, as determined by the DPPH assay, was reported by Muir (1996).Siriwardhana and Shahidi (2002) reported that whole almond seed extractscavenged 21% (at concentration at 100 ppm) and 73% (at 200 ppm) of theDPPH radical. In the latter study, a 100% scavenging activity of the DPPHradical was observed for brown almond skin and green shell extracts at 100-and 200-ppm concentrations, respectively. Dueñas et al. (2003b) reported thescavenging activity of the DPPH radical for the proanthocyanidins present inmocan fruits.

Figure 3 displays the reduction power of the acetone extract of adzukibean and its fractions. The results indicate that fraction II exhibited a greaterreduction power than the crude extract and fraction I. The reducing power ofthe adzuki bean tannin fraction was similar to that reported by Amarowiczet al. (2000b) for tannins of canola hulls. In a cited study, the reduction power

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FIG. 1. ANTIOXIDANT ACTIVITY OF AN ACETONIC ADZUKI BEAN EXTRACT AND ITSFRACTIONS IN A b-CAROTENE-LINOLEATE MODEL SYSTEM, AS MEASURED BY

CHANGES IN THE ABSORBANCE AT 470 nm


of tannins from beach pea, evening primrose and faba bean was more than twotimes greater than that of fraction II reported in this study. The same relationbetween the reducing power of extracts and its low-molecular-weight pheno-lics and tannin fractions was observed for vetch (Amarowicz et al. 2008a) andpea (Amarowicz and Troszynska 2003).

Figure 4 shows the HPLC chromatogram of the separation of phenolicconstituents from the adzuki bean extract. Table 1 presents the wavelength ofmaximum UV absorption and molecular ions from HPLC-MS.

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FIG. 2. SCAVENGING EFFECT OF CRUDE PHENOLIC EXTRACTS FROM AN ACETONICADZUKI BEAN EXTRACT AND ITS FRACTIONS ON THE

2,2-DIPHENYL-1-PICRYLHYDRAZYL RADICAL, AS MEASURED BY CHANGES INABSORBANCE AT 517 nm


There were 29 compounds identified in the adzuki bean crude extractusing HPLC-PAD (Fig. 4, Table 2). They belong to different classes of phe-nolic compounds. In addition, tryptophan and tryptohol were found. Amongphenolic acids and derivatives, protocatechuic acid and aldehyde, transp-coumaric acid and its malonyl ester were identified. Quercetin, quercetin3-O-arabinglucoside, quercetin 3-O-rutinoside, quercetin 3-O-galactoside,

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FIG. 3. REDUCING POWER OF THE CRUDE EXTRACT AND ITS FRACTIONS, ASMEASURED BY CHANGES IN ABSORBANCE AT 700 nm

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FIG. 4. HPLC CHROMATOGRAPHIC PROFILE OF THE PHENOLIC COMPOUNDSDETERMINED IN THE ADZUKI BEAN CRUDE EXTRACT

For peak identification, see Table 2.


quercetin 3-O-glucoside, myricetin 3-O-rhamnoside and kaempferol 3-O-rutinoside were found as the representative flavonols.

In the ESI-MS spectra of compounds giving peaks 2 and 6, there was anegative molecular ion [M-H]- at m/z 451.1 recorded, corresponding to amonomer of flavanol, catechin or epicatechin, linked to glucose, and a frag-ment ion at m/z 289, corresponding to catechin or epicatechin. These com-pounds were identified as catechin glucoside (2) and epicatechin glucoside (6),respectively, and these agree with the sequence of the retention time of theircorresponding aglucones.

The ESI-MS spectra of compounds corresponding to peaks 4, 16, 19 and28 showed a negative molecular ion [M-H]- at m/z 577.1 originating from a

TABLE 2.CHEMICAL COMPOUNDS IDENTIFIED BY HPLC-PAD-MS IN THE ANALYZED ADZUKI

BEAN CRUDE EXTRACT

Peak/compoundnumber

lmax (nm) [M-H]- (m/z) Ion fragments(m/z)

Compounds

1 254, 294 153 Protocatechuic acid2 279 451.1 289.1 Catechin glucoside3 280, 311 137.1 Protocatechuic aldehyde4 279 577.1 289.1 Procyanidin dimer (1)5 277 203.1 Tryptophan6 286 451.1 289.1 Epicatechin glucoside7 284 303.1 Dihydroquercetin derivative8 279 865.2 289.1 Procyanidin trimer (1)9 279 865.2 Procyanidin trimer (2)

10 309 278.0 163.1 trans-p-Coumaroyl malic acid11 284 465.1 303.1 Dihydroquercetin hexose (1)12 284 465.1 303.1 Dihydroquercetin hexose (2)13 275 457.1 Epigallocatechin gallate14 279 289.1, 169.1 Procyanidin gallate15 292 435.1 Tetrahydroxydihydrochalcone

glycoside16 279 577.0 289.1 Procyanidin dimer (2)17 279 289.1 Epicatechin18 314 163.1 trans-p-Coumaric acid19 279 577.0 289.1 Procyanidin dimer (3)20 280 160.1 Tryptohol21 289 303.1 Dihydroquercetin22 256, 357 595.1 301.1 Quercetin arabinglucoside23 255, 357 463.1 317.1 Myricetin rhamnoside24 256, 353 609.1 301.1 Quercetin rutinoside25 256, 357 463.1 301.1 Quercetin galactoside26 256, 357 461.1 301.1 Quercetin glucoside27 265, 346 593.1 Kaempferol rutinoside28 279 577.1 289.1 Procyanidin dimer (4)29 256, 270 301.1 Quercetin


procyanidin dimmer, and a negative ion at m/z 289.1, which corresponds tocatechin or epicatechin. These compounds were identified as procyanidindimers.

Compounds 8 and 9 were characterized by a negative molecular ion[M-H]- at m/z 865.2 corresponding to a procyanidin trimer, and a fragment atm/z 289.1 from a monomer, and they were identified as procyanidin trimers.

The UV spectrum of compound 10 with a maximum at 309 nm wassimilar to that of p-coumaric acid. In its ESI-MS spectrum, a negative molecu-lar ion [M-H]- at m/z 278.0 was recorded, which corresponds to p-coumaricacid linked to a malic acid, and one fragment ion [M-H]- at m/z 163.0,corresponding to a p-coumaric acid residue. This compound was identified astrans p-coumaroyl-malic acid. The presence of this compound has beenreported before in the cotyledon of lentil (Dueñas et al. 2002) and pea (Dueñaset al. 2004).

Four dihydroflavonols were identified in the adzuki bean extract (com-pounds 7, 11, 12, 21) (Table 2). Their UV spectra showed maxima at 284 nm.In the analysis by high-performance liquid chromatography with electronspray ionization mass spectrometry detector (HPLC-ESI-MS), these com-pounds showed a fragment ion at m/z 303.1 corresponding to dihydroquerce-tin. In the case of compounds 11 and 12, a negative molecular ion [M-H]- atm/z 465.1 due to dihydroquercetin linked to a hexose was recorded, and it hasbeen identified as dihydroquercetin hexose. Peak 21, with a lmax of 289 nm,was identified as dihydroquercetin.

Compound 13 was identified as epigallocatechin gallate by comparingretention time and UV spectrum with those of a standard and confirmed by thepresence of the negative molecular ion [M-H]- at m/z 457.1 in an ESI-MSspectrum. Peak 14 showed a lmax of 279 nm in the UV spectrum, whichcorresponds to a procyanidin, and in the HPLC-MS (ESI) analysis, presentedas a negative molecular ion [M-H]- at m/z of 289.1 from catechin or epicat-echin and at m/z 169.1 from gallic acid, this peak was identified as a procya-nidin gallate.

A compound giving a peak with retention time of 28.8 min had a UVspectrum similar to that of phloridzin (2′,4,4′,6′-tetrahydroxydihydrochalcone-2′-O-b-glycoside), and in the HPLC-MS (ESI) analysis, it presented a negativemolecular ion [M-H]- at m/z 435.1, which may correspond to a tetrahydroxy-dihydrochalcone linked to a hexose. This compound was identified as a gly-coside of tetrahydroxydihydrochalcone (Table 2).

Tryptophan was identified by comparing the retention time and UVspectrum (Table 2) with those of a standard. In the HPLC-ESI (MS) analysis,this peak presented a negative molecular ion [M-H]- at m/z 203.0 correspond-ing to tryptophan, an aromatic amino acid that was extracted under the experi-mental conditions and detected during the analysis of phenolic compounds.


The presence of this amino acid could be due to the high protein content oflegumes.

Peaks 22, 24, 25 and 26 were identified as different glycosides of quer-cetin (Table 2), and peak 29 as quercetin itself (Table 2) as determined by UVspectra and MS ions. Peaks 23 and 27 were identified in an identicalfashion as myricetin rhamnoside and kaempferol rutinoside, respectively(Table 2).

The content of individual phenolic compounds in the adzuki bean crudeextract is reported in Table 3. Catechin and epicatechin glucosides, quercetinglucoside, myricetin and procyanidin dimers were the dominant phenoliccompounds. Among phenolic acids, protocatechuic acid was found to be thedominant one. The presence of glycosides of flavones and flavonols in peas

TABLE 3.CONTENT OF INDIVIDUAL PHENOLIC COMPOUNDS IN ADZUKI BEAN CRUDE

EXTRACT

Compounds Peak/compound number* Content (mg/g)

Protocatechuic acid 1 67.6 � 4.01Protocatechuic aldehyde 3 7.71 � 0.62trans-p-Coumaric acid 18 31.3 � 1.96trans-p-Coumaroyl malic acid 10 4.57 � 0.69Epicatechin 17 25.7 � 2.06Epigallocatechin gallate 13 0.14 � 0.02Epicatechin glucoside 6 159.0 � 8.31Catechin glucoside 2 688.0 � 35.6Quercetin 29 36.2 � 1.54Quercetin rutinoside 24 38.2 � 1.52Quercetin galactoside 25 46.9 � 5.87Quercetin glucoside 26 181.0 � 9.14Quercetin arabinoglucoside 22 42.8 � 4.23Dihydroquercetin 21 1.15 � 0.07Dihydroquercetin hexose (1) 11 0.54 � 0.02Dihydroquercetin hexose (2) 12 0.48 � 0.05Dihydroquercetin derivative 7 1.35 � 0.06Myricetin rhamnoside 23 212.0 � 9.85Kaempferol rutinoside 27 38.2 � 1.52Tetrahydroxydihydrochalcone glycoside 15 0.55 � 0.08Procyanidin gallate 14 12.4 � 1.06Procyanidin dimer (1) 4 213.0 � 13.2Procyanidin dimer (2) 16 15.9 � 1.56Procyanidin dimer (3) 19 25.3 � 1.53Procyanidin dimer (4) 28 16.0 � 0.89Procyanidin trimer (1) 8 41.8 � 1.11Procyanidin trimer (2) 9 42.4 � 1.09

* Number of peaks corresponding to Fig. 4.


cotyledon was reported by Dueñas et al. (2006). Cowpeas were characterizedby a high content of quercetin 3-O-glucosides and myricetin 3-O-glucosides(Dueñas et al. 2005). The presence of procyanidins B-1 and B-3 was describedby Ariga and Hamano (1990) and Ariga et al. (1988). Phenolic acids (caffeic,o-coumaric, ferulic, sinapic) were found in extracts of pea, red bean, whitebean and vetch (Amarowicz and Troszynska 2003).

REFERENCES

ALSALVAR, A., KARAMAC, M., AMAROWICZ, R. and SHAHIDI, F.2006. Antioxidant and antiradical activities in extracts of hazelnut kernel(Corylus avellana L.) and hazelnut green leafy cover. J. Agric. FoodChem. 54, 4826–4832.

AMAROWICZ, R. and RAAB, B. 1997. Antioxidative activity of leguminousseed extracts evaluated by chemiluminescence methods. Z. Naturforsch.52c, 709–712.

AMAROWICZ, R. and SHAHIDI, F. 1995. A rapid chromatographic methodfor separation of individual catechins from green tea. Food Res. Int. 29,71–76.

AMAROWICZ, R. and TROSZYNSKA, A. 2003. Antioxidant activity ofextract of pea and its fractions of low molecular phenolics and tannins.Pol. J. Food Nutr. Sci. 12/53 SI 1, 10–15.

AMAROWICZ, R. and TROSZYNSKA, A. 2004. Antioxidant and antiradicalactivity of extracts of phnolic compounds from red bean. Czech J. FoodSci. 22, 206–208.

AMAROWICZ, R., PISKUŁA, M., HONKE, J., RUDNICKA, B.,TROSZYNSKA, A. and KOZŁOWSKA, H. 1995. Extraction of phe-nolic compounds from lentil seeds (Lens culinaris) with various solvents.Pol. J. Food Nutr. Sci. 4/45, 53–62.

AMAROWICZ, R., TROSZYNSKA, A., KARAMAC, M. andKOZŁOWSKA, H. 1996a. Antioxidative properties of legumeseed extracts. In Agri-Food Quality. An Inter-disciplinaryApproach (G.R. Fenwick, C. Hedley, R.L. Richards and S. Khokahr,eds.) pp. 376–379, The Royal Society of Chemistry, Cambridge,U.K.

AMAROWICZ, R., KARAMAC, M., KMITA-GŁAZEWSKA, H.,TROSZYNSKA, A. and KOZŁOWSKA, H. 1996b. Antioxidant activityof phenolic fractions of everlasting pea, faba bean and broad bean.J. Food Lipids 3, 199–211.

AMAROWICZ, R., NACZK, M. and SHAHIDI, F. 2000a. Antioxidant activ-ity of crude phenolic extracts of canola/rapeseed hulls. J. Am. Oil Chem.Soc. 77, 957–961.


AMAROWICZ, R., NACZK, M., ZADERNOWSKI, R. and SHAHIDI, F.2000b. Antioxi- dant activity of condensed tannins of beach pea, canolahulls, evening primrose, and faba beans. J. Food Lipids 7, 199–211.

AMAROWICZ, R., KARAMAC, M. and WEIDNER, S. 2001. Antioxidativeactivity of phe-phenolic fractions of pea (Pisum sativum). Czech J. FoodSci. 19, 139–142.

AMAROWICZ, R., KARAMAC, M. and SHAHIDI, F. 2003. Antioxidantactivity of phenolic fractions of lentil (Lens culinaris). J. Food Lipids 10,1–10.

AMAROWICZ, R., TROSZYNSKA, A., BARYŁKO-PIKIELNA, N. andSHAHIDI, F. 2004a. Extracts of polyphenolics from legume seeds –correlation between their total antioxidant activity, total phenolicscontent, tannins content and astringency. J. Food Lipids 11, 278–286.

AMAROWICZ, R., PEGG, R.B., RAHIMI-MOGHADDAM, P., BARL, B.and WEIL, J.A. 2004b. Free-radical scavenging capacity and antioxidantactivity of selected plant species from the Canadian prairies. Food Chem.84, 551–562.

AMAROWICZ, R., KARAMAC, M., WEIDNER, S. and SHAHIDI, F.2004c. Antioxidative activity of phenolic fractions of white bean(Phaseolus vulgaris L.). J. Food Lipids 11, 165–177.

AMAROWICZ, R., TROSZYNSKA, A. and PEGG, R.B. 2008a. Antioxidantactivity of extract of phenolic compounds from vetch (Vicia sativum).Fitoterapia (in press).

AMAROWICZ, R., ESTRELLA, I., ERNÁNDEZ, T. and TROSZYNSKA, A.2008b. Antioxidant activity of extract of phenolic compounds from redlentil. Food Res. Int. (in press).

ARIGA, T. and HAMANO, M. 1990. Radical scavenging action and its modein procyanidins B-1 and B-3 from adzuki beans to peroxyl radicals.Agric. Biol. Chem. 54, 2499–2504.

ARIGA, T., KOSHIYAMA, I. and FUKUSHIMA, D. 1988. Antioxidativeproperties of procyanidins B-1 and B-3 from adzuki beans in aqueoussystem. Agric. Biol. Chem. 52, 2717–2722.

BERGER, M., KÜCHLER, T., MAASSEN, A., BUSCH-STOCKFISCH, M.and STEINHART, H. 2007. Correlation of ingredients with sensoryattributes in green beans and peas under different storage conditions.Food Chem. (in press).

BETANCUR-ANCONA, D., PERANZA-MERCADO, G., MOGUEL-ORDOÑEZ, Y. and FUERTES-BLANCO, S. 2004. Physicochemicalcharacterization of lima bean (Phaseolus lunatus) and jack bean(Canavalia ensiformis) fibrous residues. Food Chem. 84, 287–295.

CARBONARO, M., VIRGILI, F. and CARNOVALE, E. 1996. Evidence forprotein-tannin interaction in legumes: Implications in the antioxidant


properties of faba bean tannins. Lebensm.-Wiss. Technol. 29, 743–750.

CHAVAN, U.D., AMAROWICZ, R. and SHAHIDI, F. 1999. Antioxidantactivity of phenolic fractions of beach pea (Lathyrus maritimus L). J.Food Lipids 6, 1–11.

DE MEJÍA, E.G., CASTAÍO-TOSTADO, E. and LOARCA-PIÑA, G. 1999.Antimutagenic effects of natural phenolic compounds in beans. Mutat.Reas. 441, 1–9.

DE PASCUALE, T.S., SANTOS-BUELGA, C. and RIVAS-GONZALO, J.C.2000. Quantitative analysis of flavan 3-ols in Spanish foodsruffs andbeverages. J. Agric. Food Chem. 48, 5331–5337.

DUEÑAS, M., ESTRELLA, I. and HERNÁNDEZ, T. 2002. Phenolic com-position of the cotyledon and the seed coat of lentil (Lens culinaris L).Eur. Food Res. Technol. 215, 478–483.

DUEÑAS, M., SUN, B.A., HERNÁNDEZ, T., ESTRELLA, I. and SPAR-ANGER, I. 2003a. Proanthocyanidins composition in the seed coat oflentils (Lens culinaris L). J. Agric. Food Chem. 51, 7999–8004.

DUEÑAS, M., HERNÁNDEZ, T. and ESTRELLA, I. 2003b. Phenolic com-position and antioxidant activity of the mocan seeds (Visnea mocaneraL.f). Food Chem. 82/3, 373–379.

DUEÑAS, M., ESTRELLA, I. and HERNÁNDEZ, T. 2004. Occurrence ofphenolic compounds in the seed coat and the cotyledon of pea (Pisumsativum L). Eur. Food Res. Technol. 219, 116–123.

DUEÑAS, M., FERNÁNDEZ, M.L., HERNÁNDEZ, T., ESTRELLA, I. andMUÑOZ, R. 2005. Bioactive phenolic compounds of cowpeas (Vignasinensis L.). Modifications by fermentation with natural microflora andwith Lactobacillus specie. J. Sci. Food Agric. 85, 297–304.

DUEÑAS, M., ESTRELLA, I. and HERNÁNDEZ, T. 2006. Assessmentin vitro antioxidant capacity of the seed coat and the cotyledon oflegumes in relation to their phenolic contents. Food Chem. 98, 95–103.

HALLIWELL, B., GUTTERIDGE, J.M.C. and CROSS, C.E. 1992. Freeradicals, antioxi-dants, and human disease; where are we now? J. Lab.Clin. Med. 119, 598–619.

HEMPEL, J. and BOHM, H. 1996. Quality and quantity of prevailing fla-vonoid glycosides of yellow and green French beans (Phaseolus vulgarisL). J. Agric. Food Chem. 44, 2114–2116.

KARAMAC, M., AMAROWICZ, R., WEIDNER, S. and SHAHIDI, S. 2004.Antioxidant activity of phenolioc fractions of white bean (Phaseolusvulgaris). J. Food Lipids 11, 165–177.

LÓPEZ-AMORÓS, M.L., HERNÁNDEZ, T. and ESTRELLA, I. 2006. Effectof germination on legume phenolic compounds and their antioxidantactivity. J. Food Compost. Anal. 19, 277–283.


MADHUJITH, T. and SHAHIDI, F. 2005a. Antioxidant potential of pea beans(Phaseoul vulgaris L). J. Food Sci. 70, S85–S90.

MADHUJITH, T. and SHAHIDI, F. 2005b. Beans: A source of natural anti-oxidants. In Phenolic Compounds in Food and Natural Health Products(F. Shahid and C.-T. Ho, eds.) pp. 83–93, American Chemical Society,U.S.A.

MADHUJITH, T., AMAROWICZ, R. and SHAHIDI, F. 2004a. Phenoliccompounds in beans and their effects on inhibition of radical-inducedDNA damage. J. Am. Oil Chem. Soc. 81, 691–696.

MADHUJITH, T., NACZK, M. and SHAHIDI, F. 2004b. Antioxidant activityof common beans (Phaseoul vulgaris L). J. Food Lipids 11, 220–233.

MCCARTY, M. 2004. Proposal for a dietary “phytochemical index”. Med.Hypotheses 63, 813–817.

MILLER, H.E. 1971. A simplified method for evaluation of antioxidant. J. Am.Oil Chem. Soc. 45, 91.

MUIR, A.D. 1996. Antioxidative activity of condensed tannins. In NaturalAntioxidants. Chemistry, Health Effects, and Applications (F. Shahidi,ed.) pp. 204–212, AOCS Press, Champaign, IL.

NACZK, M. and SHAHIDI, F. 1989. The effect of methanol – ammonia –water treatment on the content of phenolic acids of canola. Food Chem.31, 159–164.

ONYENEHO, S.N. and HETTIARACHCHY, N.S. 1991. Effect of navy beanhull extract on the oxidative stability of soy and sunflower oils. J. Agric.Food Chem. 39, 1701–1704.

OYAIZU, M. 1986. Studies on products of browning reaction: Antioxidativeactivities of products of browning reaction prepared from glucosamine.Jpn. J. Nutr. 44, 307–315.

PRICE, M.L., VAN SCOYOC, S. and BUTLER, L.G. 1978. A critical evalu-ation of the vanillin reaction as an assay for tannin in sorghum grain.J. Agric. Food Chem. 26, 1214–1218.

RAAB, B., HEMPEL, J. and BOHM, H. 1996. Antioxidative and antigeno-toxic properties of flavonoids prevailing in vegetable. In Agri-FoodQuality. An Interdisciplinary Approach (G.R. Fenwick, C. Hedley, R.L.Richards and S. Khokahr, eds.) pp. 369–371, The Royal Society ofChemistry, Cambridge, U.K.

RANDHIR, R. and SHETTY, K. 2004. Microwave-induced stimulation ofL-DOPA, phenolics and antioxidant activity in faba bean (Ficia faba) forParkinson’s diet. Process Biochem. 39, 1775–1784.

RANDHIR, R., LIN, Y.-T. and SHETTY, K. 2004. Stimulation of phenolics,antioxidant and antimicrobial activities in dark germinated mungbean sprouts in respect to peptide and phytochemical elicitors. ProcessBiochem. 39, 637–646.


ROCHA-GUZMÁN, N.E., GONZÁLEZ-LAREDO, R.F., IBARRA-PÉREZ,F.J., NAVA-BERÚMEN, C.A. and GALLEGOS-INFANTE, J.-A. 2007a.Effect of cooking on the antioxidant activity of extracts from threecommon bean (Phaseolus vulgaris L.) cultivars. Food Chem. 100, 31–35.

ROCHA-GUZMÁN, N.E., HERZOG, A., GONZÁLEZ-LAREDO,R.F., IBARRA-PÉREZ, F.J., ZAMBRANO-GALVÁN, G. andGALLEGOS-INFANTE, J.-A. 2007b. Antioxidant and antimutagenicactivity of phenolic compounds in three different colour groups ofcommon bean cultivars (Phaseolus vulgaris L). Food Chem. 100, 31–35.

SHAHIDI, F. 2000. Antioxidants in food and food antioxidants. Nahrung 44,158–163.

SIRIWARDHANA, S.S.K. and SHAHIDI, F. 2002. Antiradical activity ofextracts of almond and its by-products. J. Am. Oil Chem. Soc. 79, 903–908.

STRUMEYER, D.H. and MALIN, M.J. 1975. Condensed tannins in grainsorghum: Isolation, fractionation, and characterisation. J. Agric. FoodChem. 23, 909–914.

TROSZYNSKA, A. and KUBICKA, E. 2001. Superoxide scavenging activityof seed coat extracts from legume seeds. Pol. J. Food Nutr. Sci. 10/51,55–59.

TSUDA, T., OSAWA, T., NAKAYAMA, S., KAWAKISHI, S. andOHSHIMA, K. 1993. Antioxidant activity of pea bean (Phaseolus vul-garis L.) extract. J. Am. Oil Chem. Soc. 70, 910–913.

YEN, G.-C. and CHEN, H.-Y. 1995. Antioxidant activity of various teaextracts in relation to their antimutagenicity. J. Agric. Food Chem. 43,27–32.

YOSHIKI, Y., SIRAKURA, T., OKUDA, K., OKUBO, K., SAKABE, T.,NGOYA, I. and TA-MURA, N. 1996. Hydrophilic oxygen radical scav-engers in the leguminous seeds and derived foods. In Agri-Food Quality.An Interdisciplinary Approach (G.R. Fenwick, C. Hedley, R.L. Richardsand S. Khokahr, eds.) pp. 360–365, The Royal Society of Chemistry,Cambridge, U.K.

ZHOU, K. and YU, L. 2006. Total phenolic contents and its antioxidantproperties of commonly consumed vegetables grown in Colorado.Lebensm.-Wiss. Technol. 39, 1155–1162.

ZIELINSKI, H. 2002. Peroxyl radical-trapping capacity of germinated legumeseeds. Nahrung 46, 100–104.

ZIELINSKI, H. and KOZŁOWSKA, H. 2000. Antioxidant activity and totalphenolics in selected cereal grains and their different morphologicalfractions. J. Agric. Food Chem. 48, 2008–2016.


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ANTIOXIDANT ACTIVITY OF EXTRACT OF ADZUKI BEAN AND ITS FRACTIONS