ANTIOXIDANT ACTIVITY OF EXTRACT OF ADZUKI BEAN AND ITS FRACTIONS ABSTRACT

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1 ANTIOXIDANT ACTIVITY OF EXTRACT OF ADZUKI BEAN AND ITS FRACTIONS RYSZARD AMAROWICZ 1,3, ISABEL ESTRELLA 2, TERESA HERNÁNDEZ 2 and AGNIESZKA TROSZYŃSKA 2 1 Department of Food Science Institute of Animal Reproduction and Food Research of Polish Academy of Sciences ul. Tuwima 10, Olsztyn, Poland 2 Instituto de Fermentaciones Industriales (CSIC) Juan de la Cierva, Madrid, Spain Submitted for Publication September 27, 2006 Revised 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 from the column using ethanol. Fraction II, consisting of tannins, was obtained using water/acetone (1:1; v/v) as the mobile phase. Phenolic compounds present in the crude extract and its fractions showed antioxidant and radical scavenging 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. Results of 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 vanillin method and expressed as absorbance units at 500 nm/1 g was 213. There were 29 compounds (hydroxycinnamics, procyanidins, gallates, flavonols, dihydroflavonols, dihydrochalcones) identified in the crude extract using a high-performance liquid chromatography with photodiode array and mass spectrometry detectors (HPLC-PAD-MS) method. Catechin and epicatechin glucosides, procyanidin dimers, myricetin and protocatechuic acid were the dominant phenolics in the extract. 3 Corresponding author. TEL: ; FAX: ; amaro@ pan.olsztyn.pl Journal of Food Lipids 15 (2008) All Rights Reserved. 2008, The Author(s) Journal compilation 2008, Blackwell Publishing 119

2 120 R. AMAROWICZ ET AL. PRACTICAL APPLICATIONS Results showing the antioxidant activity of adzuki bean extract and its fraction can be useful for producers of healthy foods. The tannin fraction separated from adzuki can be applied for production of natural antioxidant preparations. The results of chemical analysis are valuable for databases of food phenolic compounds. INTRODUCTION Many phytochemicals found in whole plant foods can help preserve vascular health and diminish cancer risk; direct antioxidant activity may mediate much of their benefit (Halliwell et al. 1992; McCarty 2004). Natural antioxidants are compounds that detoxify reactive oxygen species and prevent their damage to cellular macromolecules and organelles through different mechanisms (Shahidi 2000). Phenolic compounds belonging to natural antioxidants are secondary metabolites commonly found in both edible and nonedible parts of plants. Evaluation of antioxidant activity of phenolic compounds from leguminous seeds has been of interest in recent years. Antioxidant activity has been reported for extracts of legumes such as pea; white, green, red and navy beans; beach pea; lentils; everlasting pea; broad bean; faba bean; lima bean; Jack bean; vetch; adzuki bean; and cowpea (Ariga et al. 1988; Ariga and Hamano 1990; Onyeneho and Hettiarachchy 1991; Tsuda et al. 1993; Amarowicz et al. 1996a,b, 2001, 2008a,b; Carbonaro et al. 1996; Hempel and Bohm 1996; Raab et al. 1996; Yoshiki et al. 1996; Amarowicz and Raab 1997; De Mejía et al. 1999; Zieliński 2002; Amarowicz and Troszyńska 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 Shahidi 2005a,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 antiradical activities of leguminous extracts have been investigated through storage studies (Onyeneho and Hettiarachchy 1991; Madhujith et al. 2004a), a b-carotene-linoleate model system (Ariga and Hamano 1990; Amarowicz et al. 1996a,b, 2004c; Amarowicz and Troszyńska 2003; Karamać et al. 2004), an EPR spin trapping method (Yoshiki et al. 1996), enhanced chemiluminescence and photoluminescence (Raab et al. 1996; Amarowicz and Raab 1997), scavenging of 2.2 -azobis (2,4-dimethylvaleronitrite) (AMVN) radical (Ariga and Hamano 1990), 2,2 -azobis (2-amidopropane) hydrochloride (ABAP) radical (Zieliński 2002), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical (Amarowicz and Troszyńska 2003; Zhou and Yu 2006), 2,2 -azinobis-3-

3 ANTIOXIDANT ACTIVITY OF ADZUKI BEAN 121 ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radical (Amarowicz and Troszyńska 2003; Zhou and Yu 2006), superoxide anion radical (Troszyńska and Kubicka 2001; Zhou and Yu 2006), peroxyl radical (Zhou and Yu 2006), reducing power (Amarowicz and Troszyńska 2003), low-density lipoprotein (LDL) cholesterol oxidation (Madhujith and Shahidi 2005a) and Fe 2+ chelatin capacity (Madhujith and Shahidi 2005b; Zhou and Yu 2006). In recent years, evidence has been accumulating about the high antioxidant potential of tannins (Muir 1996; Amarowicz et al. 2000a,b, 2004a,b; Dueñas et al. 2003a). The high impact of tannins of leguminous seed extracts has previously been reported (Amarowicz and Troszyńska 2003; Amarowicz et al. 2004a, 2008a; Dueñas et al. 2006). The objectives of this research were to investigate the antioxidant and antiradical activities of an acetone extract of adzuki beans and its low-molecular-weight and tannin fractions. The occurrence 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 acquired from the P.O.Ch. Company (Gliwice, Poland). Butylated hydroxyanisole, b-carotene, linoleic acid, vanillin, Folin Ciocalteau s reagent, polyoxyethylenesorbitan monopalmitate (Tween 40), Sephadex LH-20 and DPPH radical were obtained from Sigma (Poznań, Poland). Protocatechuic acid, protocatechuic aldehyde, trans p-coumaric acid, (+)-catechin, ( ) epicatechin, dihydroquercetin, quercetin, quercetin 3-O-glucoside, quercetin 3-O-galactoside, quercetin 3-O-rutinoside, myricetin 3-O-rhamnoside, kaempferol 3-Orutinoside, tryptophan, 2,4,4,6 -tetrahydroxydihydrochalcone-2 -O-bglucoside (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 hexanes in a Soxhlet apparatus for 6 h. Phenolic compounds were extracted from the raw 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 in dark-colored flasks using a shaking water bath (Elpan 357, Wroclaw, Poland). The extraction was repeated twice, supernatants combined, and acetone was

4 122 R. AMAROWICZ ET AL. 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 and tannin fraction was carried out according to the method described by Strumeyer and Malin (1975). A 2-g portion of the crude extract was suspended in 20 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 eluted from the column using 1 L of 95% (v/v) ethanol. To obtain tannins (fraction II), the column was washed with 500 ml of 50% (v/v) acetone. Organic solvents were evaporated and water was removed by lyophilization. Total Phenolics The content of total phenolic compounds in extract and each fraction was estimated using the Folin Ciocalteau s phenol reagent (Naczk and Shahidi 1989). (+)-Catechin was used as a standard. Content of Tannins The content of tannins in the crude extract and its fractions was determined using the modified vanillin method assay (Price et al. 1978); results were expressed as absorbance units at 500 nm/g extract (A 500 /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 provided by the supplier; a concentration of 2 mg extracts/ml methanol was used in 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 its fractions was determined in an emulsion system using the method described by Miller (1971). Methanolic solutions (0.2 ml) containing 2 mg of crude extract or fraction I or 1 mg of fraction II were added to a series of tubes containing 5 ml of a prepared emulsion of linoleate and b-carotene stabilized with Tween 40 (Sigma). Immediately after the addition of emulsion to each

5 ANTIOXIDANT ACTIVITY OF ADZUKI BEAN 123 tube, the zero-time absorbance at 470 nm was recorded. Samples were kept in a water bath at 50C, and their absorbance values were recorded over a 120-min period 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 distilled water was mixed with 2.5 ml of 0.2 M phosphate buffer (ph 6.6) and 2.5 ml of 1% (w/v) potassium ferricyanide. The mixture was incubated at 50C for 20 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 aliquot of the upper layer was mixed with 2.5 ml of distilled water and 0.5 ml of 0.1% (w/v) FeCl 3 ; the absorbance of the mixture was read at 700 nm. Scavenging of DPPH Radical The scavenging effect of phenolics from the crude adzuki bean extract and its two fractions was monitored as described by Yen and Chen (1995). A 0.1-mL methanolic solution containing mg of extract or fraction I and mg fraction II was mixed with 2 ml of water and then added to a methanolic solution of DPPH (1-mM 0.25 ml). The mixture was vortexed for 1 min, then left to stand at room temperature for 20 min, and the absorbance of this solution was subsequently read at 517 nm. High-performance Liquid Chromatography with Photodiode Array Detector (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 quaternary pump, a photodiode-array detector 2001 (Waters, Milford, MA) and Nova-Pak C 18 ( ; 4 mm) column (Waters). The analytical conditions were those described by Dueñas et al. (2004). Two mobile phases were employed 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, % A; min, 20 10% A; min, 10 5% A; and min, 100% B. The flow rate was 1 ml/min from the beginning to 55 min and 1.2 ml/min from this point to the end. The column was re-equilibrated between injections with 10 ml of acetonitrile and 25 ml of the initial mobile phase. Detection was performed by scanning from 210 to 400 nm with an acquisition speed of 1 s. A volume of 100 ml was injected. The sample was analyzed in duplicate.

6 124 R. AMAROWICZ ET AL. High-performance Liquid Chromatography with Mass Spectrometry Detector (HPLC-MS) Analysis Mass spectra were obtained using a Hewlett Packard 1100 MSD (Palo Alto, CA) chromatograph equipped with an API source, using an electron spray ionization (ESI) interface and the conditions reported by Dueñas et al. (2004). The solvent gradient and column used were the same as those for HPLC-PAD. ESI conditions were as follows: negative mode, nitrogen was used as the nebulizing gas, 40 psi, drying gas, 10 L/min at 340C; voltage at capillary entrance, 4,000 V; and variable fragmentation voltage, 100 V (m/z 200 1,000), 250 V (m/z 1,000 2,500). Mass spectra were recorded from m/z 100 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 to those of standards (standards used are listed in Materials). Other compounds with UV spectra similar to those of hydroxycinnamates, procyanidins, gallates, flavonols, dihydroflavonols and dihydrochalcones were identified as their derivatives. Their chemical structures were confirmed by HPLC-MS (ESI). Quantification was made using the external standard method according to the maximum of absorption of each compound. The calibration curves were made by injection of different volumes of the standards from a stock solution over the concentration range observed for each compound. The calibration curves were calculated as linear regressions of peak area versus standard concentration. The trans-p-coumaric acid derivative was quantified using the calibration curve of the corresponding free phenolic acid, procyanidins using the calibration curve of (+)-catechin. Dihydrochalcone was expressed as phloridzin, and dihydroflavonols as dihydroquercetin. RESULTS AND DISCUSSION The content of total phenolics in fraction I was almost two times lower than that in the crude extract and more than three times lower than in fraction II (Table 1). A similar relation between the content of total phenolics in a crude extract and its low-molecular-weight phenolics and tannin fractions was observed in the case of pea (Amarowicz et al. 2003), red bean (Amarowicz and Troszyńska 2004), vetch (Amarowicz et al. 2008a) and red lentil (Amarowicz et al. 2008b). For Sephadex LH-20 column chromatography, the first solvent used was ethanol. Ethanol can elute some phenolics together with sugars from the column. Because sugars were dominant compounds of the crude extract,

7 ANTIOXIDANT ACTIVITY OF ADZUKI BEAN 125 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 (A 500/g) Total antioxidant activity (mmol Trolox/mg) Crude extract Fraction I Fraction II A 500/g, absorbance units at 500 nm/1 g extract. the content of phenolics in fraction I was low. A high content of total phenolics in the tannin fraction, separated from the crude extract using a Sephadex LH-20 column, was described previously for extracts of leguminous seeds such as beach pea, faba bean (Amarowicz et al. 2000b), pea (Amarowicz and Troszyńska 2003), vetch (Amarowicz et al. 2008b), red lentil (Amarowicz et al. 2008b), canola hulls and evening primrose (Amarowicz et al. 2000b). The content of tannins, expressed as absorbance value at 500 nm/g, in fraction II (213), was about two times higher than that in the crude extract (136). For the low-molecular-weight phenolic fraction, the observed vanillinpositive reaction could be due to catechin and other flavan-3-ols, which can elute from the Sephadex LH-20 column with ethanol (Amarowicz and Shahidi 1995). The presence of tannins in leguminous seeds has been reported by several 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 methanolic extracts of wheat, barley, rye and oat, Zieliński and Kozłowska (2000) reported lower values of TAA ( mmol Trolox/mg) than those obtained in this study. For extract of seed coats from pea, TAA was 3.6 mmol Trolox/mg (Troszyńska and Kubicka 2001). Vetch extract and its low-molecular-weight phenolics and tannin fractions exhibited TAA values of 0.79, 0.40 and 6.40 mmol Trolox/mg, respectively (Amarowicz et al. 2008a). The acetone extract of phenolic compounds from adzuki bean and fractions I and II exhibited antioxidant activity in a b-carotene-linoleate model system (Fig. 1). The effect of the extract on the coupled oxidation of linoleic acid and b-carotene was the highest, but the addition of the tannin fraction was only 1 mg. On the other hand, these fractions might be quickly oxidized and therefore could not be effective as an antioxidant in the emulsion model. Using the same method, similar or lower antioxidant activities were found for

8 126 R. AMAROWICZ ET AL. 0.6 BHA Control Extract Fraction I Fraction II Absorbance at 470 nm Time (min) FIG. 1. ANTIOXIDANT ACTIVITY OF AN ACETONIC ADZUKI BEAN EXTRACT AND ITS FRACTIONS IN A b-carotene-linoleate MODEL SYSTEM, AS MEASURED BY CHANGES IN THE ABSORBANCE AT 470 nm extracts of leguminous seeds such as faba bean, broad bean, lentil, beach pea and everlasting bean (Amarowicz et al. 1996a,b; Chavan et al. 1999). The strong scavenging effect of tannins separated from the adzuki bean extract was confirmed in a DPPH assay (Fig. 2). The tannin fraction exhibited antiradical activity several times higher than that of the crude extract. The low-molecular-weight phenolic fraction was a weaker scavenger of the DPPH radical. The scavenging effect of condensed tannins from beach pea, canola hulls, evening primrose and faba bean on the DPPH radical was described by Amarowicz et al. (2000b). The radical scavenging activity of condensed tannins, as determined by the DPPH assay, was reported by Muir (1996). Siriwardhana and Shahidi (2002) reported that whole almond seed extract scavenged 21% (at concentration at 100 ppm) and 73% (at 200 ppm) of the DPPH radical. In the latter study, a 100% scavenging activity of the DPPH radical was observed for brown almond skin and green shell extracts at 100- and 200-ppm concentrations, respectively. Dueñas et al. (2003b) reported the scavenging activity of the DPPH radical for the proanthocyanidins present in mocan fruits. Figure 3 displays the reduction power of the acetone extract of adzuki bean and its fractions. The results indicate that fraction II exhibited a greater reduction power than the crude extract and fraction I. The reducing power of the adzuki bean tannin fraction was similar to that reported by Amarowicz et al. (2000b) for tannins of canola hulls. In a cited study, the reduction power

9 ANTIOXIDANT ACTIVITY OF ADZUKI BEAN 127 Absorbance at 517 nm Extract Fraction I Content (mg/assay) Absorbance at 517 nm Fraction II Content (mg/assay) FIG. 2. SCAVENGING EFFECT OF CRUDE PHENOLIC EXTRACTS FROM AN ACETONIC ADZUKI BEAN EXTRACT AND ITS FRACTIONS ON THE 2,2-DIPHENYL-1-PICRYLHYDRAZYL RADICAL, AS MEASURED BY CHANGES IN ABSORBANCE AT 517 nm of tannins from beach pea, evening primrose and faba bean was more than two times greater than that of fraction II reported in this study. The same relation between the reducing power of extracts and its low-molecular-weight phenolics and tannin fractions was observed for vetch (Amarowicz et al. 2008a) and pea (Amarowicz and Troszyńska 2003). Figure 4 shows the HPLC chromatogram of the separation of phenolic constituents from the adzuki bean extract. Table 1 presents the wavelength of maximum UV absorption and molecular ions from HPLC-MS.

10 128 R. AMAROWICZ ET AL. Optical density at 700 nm Extract Fraction I Fraction II Content (mg/assay) FIG. 3. REDUCING POWER OF THE CRUDE EXTRACT AND ITS FRACTIONS, AS MEASURED BY CHANGES IN ABSORBANCE AT 700 nm AU 280 nm min FIG. 4. HPLC CHROMATOGRAPHIC PROFILE OF THE PHENOLIC COMPOUNDS DETERMINED IN THE ADZUKI BEAN CRUDE EXTRACT For peak identification, see Table 2. There were 29 compounds identified in the adzuki bean crude extract using HPLC-PAD (Fig. 4, Table 2). They belong to different classes of phenolic compounds. In addition, tryptophan and tryptohol were found. Among phenolic acids and derivatives, protocatechuic acid and aldehyde, trans p-coumaric acid and its malonyl ester were identified. Quercetin, quercetin 3-O-arabinglucoside, quercetin 3-O-rutinoside, quercetin 3-O-galactoside,

11 ANTIOXIDANT ACTIVITY OF ADZUKI BEAN 129 TABLE 2. CHEMICAL COMPOUNDS IDENTIFIED BY HPLC-PAD-MS IN THE ANALYZED ADZUKI BEAN CRUDE EXTRACT Peak/compound number l max (nm) [M-H] - (m/z) Ion fragments (m/z) Compounds 1 254, Protocatechuic acid Catechin glucoside 3 280, Protocatechuic aldehyde Procyanidin dimer (1) Tryptophan Epicatechin glucoside Dihydroquercetin derivative Procyanidin trimer (1) Procyanidin trimer (2) trans-p-coumaroyl malic acid Dihydroquercetin hexose (1) Dihydroquercetin hexose (2) Epigallocatechin gallate , Procyanidin gallate Tetrahydroxydihydrochalcone glycoside Procyanidin dimer (2) Epicatechin trans-p-coumaric acid Procyanidin dimer (3) Tryptohol Dihydroquercetin , Quercetin arabinglucoside , Myricetin rhamnoside , Quercetin rutinoside , Quercetin galactoside , Quercetin glucoside , Kaempferol rutinoside Procyanidin dimer (4) , Quercetin quercetin 3-O-glucoside, myricetin 3-O-rhamnoside and kaempferol 3-Orutinoside were found as the representative flavonols. In the ESI-MS spectra of compounds giving peaks 2 and 6, there was a negative molecular ion [M-H] - at m/z recorded, corresponding to a monomer of flavanol, catechin or epicatechin, linked to glucose, and a fragment ion at m/z 289, corresponding to catechin or epicatechin. These compounds were identified as catechin glucoside (2) and epicatechin glucoside (6), respectively, and these agree with the sequence of the retention time of their corresponding aglucones. The ESI-MS spectra of compounds corresponding to peaks 4, 16, 19 and 28 showed a negative molecular ion [M-H] - at m/z originating from a

12 130 R. AMAROWICZ ET AL. procyanidin dimmer, and a negative ion at m/z 289.1, which corresponds to catechin or epicatechin. These compounds were identified as procyanidin dimers. Compounds 8 and 9 were characterized by a negative molecular ion [M-H] - at m/z corresponding to a procyanidin trimer, and a fragment at m/z from a monomer, and they were identified as procyanidin trimers. The UV spectrum of compound 10 with a maximum at 309 nm was similar to that of p-coumaric acid. In its ESI-MS spectrum, a negative molecular ion [M-H] - at m/z was recorded, which corresponds to p-coumaric acid 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 as trans p-coumaroyl-malic acid. The presence of this compound has been reported before in the cotyledon of lentil (Dueñas et al. 2002) and pea (Dueñas et al. 2004). Four dihydroflavonols were identified in the adzuki bean extract (compounds 7, 11, 12, 21) (Table 2). Their UV spectra showed maxima at 284 nm. In the analysis by high-performance liquid chromatography with electron spray ionization mass spectrometry detector (HPLC-ESI-MS), these compounds showed a fragment ion at m/z corresponding to dihydroquercetin. In the case of compounds 11 and 12, a negative molecular ion [M-H] - at m/z due to dihydroquercetin linked to a hexose was recorded, and it has been identified as dihydroquercetin hexose. Peak 21, with a l max of 289 nm, was identified as dihydroquercetin. Compound 13 was identified as epigallocatechin gallate by comparing retention time and UV spectrum with those of a standard and confirmed by the presence of the negative molecular ion [M-H] - at m/z in an ESI-MS spectrum. Peak 14 showed a l max of 279 nm in the UV spectrum, which corresponds to a procyanidin, and in the HPLC-MS (ESI) analysis, presented as a negative molecular ion [M-H] - at m/z of from catechin or epicatechin and at m/z from gallic acid, this peak was identified as a procyanidin gallate. A compound giving a peak with retention time of 28.8 min had a UV spectrum similar to that of phloridzin (2,4,4,6 -tetrahydroxydihydrochalcone- 2 -O-b-glycoside), and in the HPLC-MS (ESI) analysis, it presented a negative molecular ion [M-H] - at m/z 435.1, which may correspond to a tetrahydroxydihydrochalcone linked to a hexose. This compound was identified as a glycoside of tetrahydroxydihydrochalcone (Table 2). Tryptophan was identified by comparing the retention time and UV spectrum (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 corresponding to tryptophan, an aromatic amino acid that was extracted under the experimental conditions and detected during the analysis of phenolic compounds.

13 ANTIOXIDANT ACTIVITY OF ADZUKI BEAN 131 TABLE 3. CONTENT OF INDIVIDUAL PHENOLIC COMPOUNDS IN ADZUKI BEAN CRUDE EXTRACT Compounds Peak/compound number* Content (mg/g) Protocatechuic acid Protocatechuic aldehyde trans-p-coumaric acid trans-p-coumaroyl malic acid Epicatechin Epigallocatechin gallate Epicatechin glucoside Catechin glucoside Quercetin Quercetin rutinoside Quercetin galactoside Quercetin glucoside Quercetin arabinoglucoside Dihydroquercetin Dihydroquercetin hexose (1) Dihydroquercetin hexose (2) Dihydroquercetin derivative Myricetin rhamnoside Kaempferol rutinoside Tetrahydroxydihydrochalcone glycoside Procyanidin gallate Procyanidin dimer (1) Procyanidin dimer (2) Procyanidin dimer (3) Procyanidin dimer (4) Procyanidin trimer (1) Procyanidin trimer (2) * Number of peaks corresponding to Fig. 4. The presence of this amino acid could be due to the high protein content of legumes. Peaks 22, 24, 25 and 26 were identified as different glycosides of quercetin (Table 2), and peak 29 as quercetin itself (Table 2) as determined by UV spectra and MS ions. Peaks 23 and 27 were identified in an identical fashion as myricetin rhamnoside and kaempferol rutinoside, respectively (Table 2). The content of individual phenolic compounds in the adzuki bean crude extract is reported in Table 3. Catechin and epicatechin glucosides, quercetin glucoside, myricetin and procyanidin dimers were the dominant phenolic compounds. Among phenolic acids, protocatechuic acid was found to be the dominant one. The presence of glycosides of flavones and flavonols in peas

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