Food Sci. Technol. Res., 16 (3), 215–224, 2010

Determination of Free and Bound Phenolic Acids, and Evaluation of Antioxidant

Activities and Total Polyphenolic Contents in Selected Pearled Barley

Atsuko YoShida1, Keisuke SoNoda1, Yoichi Nogata2, Takashi NagamiNe

2, Maki Sato3, Tomoyuki oki3,

Shunjiro haShimoto4 and Hideaki ohta

1*

1 Graduate School of Health and Nutrition Sciences, Nakamura Gakuen University, 5-7-1 Befu, Johnan-ku, Fukuoka 814-0198, Japan2 National Agricultural Research Center for Western Region, National Agriculture & Food Research Organization, 6-12-1 Nishifukatsu, Fukuyama, Hiroshima 721-8514, Japan

3 National Agricultural Research Center for Kyushu Okinawa Region, National Agriculture & Food Research Organization, 2421 Suya, Koshi, Kumamoto 861-1192, Japan

4 Department of Food and Nutrition, Nakamura Gakuen College, 5-7-1 Befu, Johnan-ku, Fukuoka 814-0198 Japan

Received December 11, 2009; Accepted January 18, 2010

The content of insoluble bound phenolic acids in pearled barley was determined by an analytical sys-tem consisting of alkaline hydrolysis extraction, high-performance liquid chromatographic separation and electrochemical detection. Insoluble bound phenolic acids in five pearled cultivars and fifteen breeding lines of barley comprised ferulic acid (4.3-34.2 mg/100 g dry matter), sinapic acid (0.025-0.445 mg/100 g), caffeic acid (0.002-0.016 mg/100 g). Soluble free polyphenols comprised procyanidins (12.2-80.3 mg/100 g), catechin (0.1-28.2 mg/100 g), and total pholyphenol comprised 152.4-324.0 mg-gallic acid equivalents/100 g. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity for the barley samples ranged from 403 to 1501 μmol-Trolox equivalents/100 g whereas those of oxygen radical absorbance capacity (ORAC) ranged from 1050 to 3816 μmol-Trolox equivalents/100 g. High correlation (0.980, p<0.01) was found be-tween the DPPH and ORAC assays. Total polyphenol contents positively correlated with the DPPH (0.875, p<0.01) and ORAC (0.881, p<0.01) assays. The correlation coefficient between insoluble bound phenolic acids and total polyphenol contents was higher than that between soluble free polyphenols and total poly-phenol contents. Taken together, the insoluble bound phenolic acids appear to greately contributed to the antioxidant activity in pearled barley.

Keywords: barley, antioxidant, insoluble bound phenolic acid, DPPH, ORAC

*To whom correspondence should be addressed.E-mail: hohta@nakamura.ac.jp

IntroductionCancer mortality rates due to gastric and uterine cancer

are decreasing in Japan, while the cancer mortality rates due to lung, colon and breast cancer are on the rise (Rama-rathnam et al., 1997). The steady decline in the incidence of gastric cancer in Japan is attributed to a change in eating habits from the traditional Japanese diet to the Western diet. Epidemiological studies indicate that a high consumption of whole cereal products reduce the risk of cardiovascular diseases and certain cancers (World Cancer Research Fund, 1997; Joint WHO/FAO Expert Consultation, 2003). These

effects have been ascribed to dietary fibers and phenolic compounds, including procyanidins (Oki et al., 2002) and cinnamic acids (Bonoli, 2004). Phenolic compounds have been shown to have strong antioxidant activities in vitro and in vivo associated with their ability to scavenge free radicals to break radical chain reactions and to chelate metals. A wide range of antioxidant components with a phenolic structure such as benzoic and cinnamic derivatives, flavonols, chal-cones, and flavones (Hernanz et al., 2001; McMurroufh & Madigan, 1996; Goupy et al., 1999) have been found in both their free and bound forms in cereals. The major free pheno-lic compounds include procyanidins and flavonoids and the major bound phenolic compounds include hydroxycinnamic acid derivatives such as caffeic, p-coumaric and ferulic acids

a DGU-14A degasser, a SCL-10A system controller, an SIL-10AXL auto injector, a CTO-10A column oven, a GL Sci-ences ED 623 electrochemical detector and a Fujitsu personal computer equipped with a Shimadzu LC work station (class-VP) (Kyoto, Japan). ECD was obtained from GL Science Ind. Co. (Tokyo, Japan). The HPLC instruments were purchased from Shimadzu Co.

Materials The five cultivars (Pirkka, Elrose, Stirling, Sukai golden, and Scarlett) and 15 breeding lines of barley (Kanto kawa 87, Kanto kawa 88, Touzan kawa 107, Kanto nijo 40, Kanto nijo 41, Kankei n551, Kankei n552, Kankei n553, Kankei n554, Daikei HQ10, Daikei HL9-2-6, BF 99-1, R 2815, M 737, and RISO 1508) used in the present study (Table 1) were obtained from the National Agricultural Re-search Center for Western Region (National Agriculture & Food Research Organization, Hiroshima, Japan). For the ex-perimental preparations, barley samples were cleaned of ex-traneous materials and their outer kernel layers were removed by 60% weight in a laboratory-scale pearlest (10 g capacity, Kett Inc., Tokyo, Japan). The pearled samples were ground using an ultra centrifugal mill (ZM200; Retsch GmbH, Haan, Germany) with a 0.5-mm stainless steel screen at ambient temperature, and then stored at −20℃.

Extraction from pearled barley samples for determination of insoluble bound phenolic acids Ground barley powder (0.5 g) was extracted by stirring for 30 min with 15 mL of n-hexane and 80% ethanol, and the supernatant was removed by centrifugation at 4,000 g for 15 min; this procedure was repeated four times. The residue was hydrolyzed with 1.0 M NaOH (2 × 30 mL, 2 h each) at ambient temperature with stirring under nitrogen gas (Tain et al., 2004). After alkaline hydrolysis, the clear supernatants were pooled, acidified with 4.0 M HCl to pH 1.0, and then extracted twice with ethyl ac-etate (150 mL each). The ethyl acetate fractions were evapo-rated to dryness, and phenolic acids were dissolved in 2 mL of 15% methanol. All samples were filtered through a 0.45-μm pore size syringe-driven filter (Advantec Toyo, Tokyo, Japan) prior to injection into HPLC (Tain et al., 2004).

Extraction from pearled barley samples for determination of soluble free phenolic acids and antioxidant activities Ground barley powder (0.5 g) was extracted by an acceler-ated solvent extractor (Type ASE-200; Dionex Co., USA) with n-hexane/dichloromethane (1:1 v/v) and acetone/water/acetic acid (70:29.5:0.5, v/v) according to the method of Wu et al. (2004). The fraction of acetone/water/acetic acid was used for examining the antioxidant activity and total polyphe-nol content (Oki et al., 2002 & 2009). For determination of free phenolic acids, an aliquot (1 mL) of the acetone/water/acetic acid fraction was evaporated to dryness under nitrogen at 40℃ and dissolved in 0.5 mL of methanol for spectropho-

which link to the cell wall by an ester bond (Waldron et al., 1996; Renger et al., 2000; Sun et al., 2002; Bonoli et al., 2004).

Due to their valuable dietary fiber, barley grains are widely used not only in soups, stews and barley bread, but also mixed with rice and steamed, as is common in Japan. Barley meal and its components are also used in health foods (i.e., paste baked products) because they contain bioactive components including β-glucans (Marconi et al., 2000) and tocols (Peterson, 1994). Several studies on barley phenolic compounds and their antioxidant activities have been report-ed (Millard and Berset, 1995; Maillard et al., 1996; Goupy et al., 1999; Duh et al., 2001). Recently, the radical scaveng-ing of pearled barley extract of two barley cultivars has been determined by measuring radical scavenging efficacy using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC), with phenolic acids as the rad-ical scavenging compounds (Madhujith et al., 2006; Terrence and Fereldoon, 2009). However, few studies have reported the antioxidant properties and phenolic acids of barley culti-vated in Japan.

In this study, we developed an analytical method to de-termine bound phenolic acids in cereals. We examined the contents of soluble free and insoluble bound phenolic acids and evaluated antioxidant activity in pearled barley cultivated in Japan to analyze the correlation between antioxidant activ-ity, the content of total polyphenol, soluble free polyphenols, and insoluble bound phenolic acids such as caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid.

Materials and MethodsReagents 6-Hydroxy-2,5,7,8-tetramethylchroman-2-

carboxylic acid (Trolox), catechin, p-coumaric acid, ferulic acid, caffeic acid, and sinapic acid and fluorescein (Na salt) were purchased from Sigma-Aldrich Co. (Tokyo, Japan). Disodium ethylenediaminetetraacetate (EDTA) and 2-mor-pholinoethanesulfonic acid (MES) were obtained from Dojin Chemical Co. (Kumamoto, Japan). DPPH and other reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan) and used without further purification. Distilled water from a Milli-Q water purification system (Millipore Japan Co., Tokyo, Japan) was used for all solutions, dilutions, and the mobile phase.

For the ORAC assay, 2,2’-azobis(2-amidinopropane)dihydrochloride (AAPH) (Wako Pure Chemical Industries, Osaka, Japan) was used.

Apparatus The high-performance liquid chromatog-raphy (HPLC) system consisted of an LC-10AT pump for liquid chromatography, a COSMOSIL 5C18-AR-II column (250 mm × 4.6 mm i.d.; Nacalai Tesque Inc., Kyoto, Japan),

a.�YoShida�et al.216

μL), and MES buffer (pH 6.0, 50 μL) were added to a 96-well microplate (Falcon Co., Tokyo, Japan). The reaction with ethanol was initiated by the addition of 800 μM DPPH (50 μL) and allowed to stand for 20 min at 30℃, before measuring absorbance at 520 nm (Multiscan JX, Thermo Fisher Scientific Co., Kanagawa, Japan). The DPPH radical scavenging activity was estimated from the decrease in ab-sorbance and expressed as micromoles of Trolox equivalents (TE) per 100 grams of dry matter (μmol-TE/100 g) based on a standard Trolox curve. In this assay, catechin, one of the constituents of procyanidins, exhibited 1.53 times higher DPPH scavenging activity than Trolox on a molar basis.

ORAC assay Antioxidant activity in the barley extract was measured using the ORAC assay according to the meth-od of Guillermo and Trust (2009) with minor modifications as reported by Oki et al. (2009). The assay is based on the principle that antioxidant compounds present in the sample will inhibit the decay in fluorescence intensity of a fluores-cent probe (fluorescein) after mixing with AAPH (a free radical generator that acts as an oxidizing agent). Antioxidant activity was measured and calculated using a automated

tometric determination of procyanidins (vanillin assay). An-other aliquot (1 mL) of the acetone/water/acetic acid fraction was evaporated to dryness in a similar manner and dissolved in 0.5 mL of 15% methanol for HPLC analysis of catechin. Samples were filtered through a 0.45-μm pore size syringe-driven filter prior to injection into HPLC.

HPLC determination of soluble free and insoluble bound phenolic acids Sample (20 μL) was injected into the HPLC system for electrochemical detection at +500 mV. As the mo-bile phase, a mixture of 100 mM phosphate buffer (pH 3.1) containing EDTA (10 mg/L), sodium dodecyl sulfate (10 mg/L), and 10% acetonitrile was eluted at a flow rate of 1.0 mL/min at 35℃. Catechin, p-coumaric acid, caffeic acid, ferulic acid and sinapic acid were identified by comparing retention times with those of standard solutions. The concentration of each compound was calculated from its integrated peak area and that of the standard.

DPPH radical scavenging activity assay The DPPH radical scavenging activity of the pearled barley extract was examined by a previously described method (Oki et al., 2002). Briefly, the sample solution (50 μL), 30% acetone (50

Antioxidant Activities and Phenolic Acids of Barley

Table 1. Pearled barley samples investigated in this study.

Sample no. Sample name Cultivar orbreeding line Source Hulled or

naked barley

1 Pirkka Cultivar Finland Hulled2 Kanto kawa 87 Breeding line Tsukuba (Japan) Hulled3 Kanto kawa 88 Breeding line Tsukuba (Japan) Hulled4 Touzan kawa 107 Breeding line Nagano (Japan) Hulled5 Kanto nijo 40 Breeding line Tsukuba (Japan) Hulled6 Kanto nijo 41 Breeding line Tsukuba (Japan) Hulled7 Kankei n551 Breeding line Tsukuba (Japan) Naked8 Kankei n552 Breeding line Tsukuba (Japan) Naked9 Kankei n553 Breeding line Tsukuba (Japan) Naked10 Kankei n554 Breeding line Tsukuba (Japan) Naked11 Daikei HQ10 Breeding line Tochigi (Japan) Hulled12 BF 99-1 Breeding line Tochigi (Japan) Hulled13 Daikei HL9-2-6 Breeding line Tochigi (Japan) Hulled14 R 2815 Breeding line Kagawa (Japan) Hulled15 Elrose Cultivar Canada Hulled16 Stirling Cultivar Australia Hulled17 M 737 Breeding line Canada Hulled18 Sukai golden Cultivar Tochigi (Japan) Hulled19 Scarlett Cultivar Canada Hulled20 RISO 1508 Breeding line Sweden Hulled

217

vanillin in methanol and 750 μL of 9.0 M H2SO4 in methanol were added to 300 μL of sample solution dissolved in metha-nol. The mixture was held for 15 min at 30℃, and absor-bance was measured at 500 nm. The procyanidin content was expressed as milligrams of catechin equivalents (Oki et al., 2002).

Statistical analysis All tests were conducted in tripli-cate. Data are reported as means ± standard deviation. The Pearson correlation analysis was performed using SPSS software (version 17.0 for Windows, SPSS Inc., Chicago, IL, USA) to determine the correlations among the mean antioxi-dant activities, total polyphenol contents and free and bound phenolic acids.

Results and DiscussionOptimization of HPLC conditions To optimize the

separation of hydroxycinnamic compounds, caffeic acid, p-coumaric acid, ferulic acid and sinapic acid, we used a COSMOSIL 5C18-AR-II column with the mobile phase, 100 mM phosphate buffer (pH 3.1) containing EDTA (10 mg/L), sodium dodecyl sulfate (10 mg/L) and 10% acetonitrile.

Figure 1 shows the chromatograms obtained for hy-droxycinnamic acid derivative standards and barley extract. A sharp peak was observed at 14.5 min for caffeic acid (1), 28.2 min for p-coumaric acid (2), 39.6 min for ferulic acid (3) and 43.3 min for sinapic acid (4). To determine the relation-ship between the amount of bound hydroxycinnamic acid

microplate pipetting system that automatically transferred ORAC reagents into a 96-well microplate, and a Synergy HT with TRF & Injector microplate fluorescence reader (Bio-Tek Instruments Inc., Tokyo, Japan) controlled by KC4 software. The latter instrument was equipped with fluorescence filters with an excitation wavelength of 485 nm and an emission wavelength of 520 nm in order to measure changes in fluo-rescence of fluorescein under controlled temperature condi-tions (37℃). The ORAC values of 20 pearled barley samples were also determined and expressed as μmol-TE/100 g.

Total polyphenol content assay The total polyphenol content of the barley extract was determined according to the Folin-Ciocalteu spectrophotometric method (Singleton & Rossi, 1965) with some modifications. Briefly, 1 mL of 2-fold diluted barley extract was mixed with 5 mL of 10-fold diluted Folin-Ciocalteu’s phenol reagent. After incubat-ing for 5 min, 4 mL of 7.5% Na2CO3 solution and deionized water were added to obtain a final volume of 10 mL, and then the solution was further incubated for 1 h at ambient temperature. Absorbance was determined to be 765 nm. The measurement was compared to a standard curve of gallic acid (GA) solution, and the total phenolic content was expressed as milligrams of gallic acid equivalents (GAE) per 100 grams of dry matter (mg-GAE/100 g).

Procyanidin determination (vanillin assay) Procyani-dins were determined using vanillin and H2SO4 according to the method of Sun et al. (1998). Briefly, 750 μL of 1.0%

Fig. 1. HPLC analysis of hydroxycinnamic acid derivative standards and barley extract.

A: Standards (0.1 mg/mL each): caffeic acid (1), p-coumaric acid (2), ferulic acid (3) and sinapic acid (4). B: Barley cultivar: Kankei n551. HPLC conditions: mobile phase, 100 mM phosphate buffer (pH 3.1) containing EDTA (10 mg/L), sodium do-decyl sulfate (10 mg/L) and 10% acetonitrile; flow rate, 1.0 mL/min; column temperature, 35℃; electrochemical detection, +500 mV; injection volume, 20 µL.

a.�YoShida�et al.

20 40

20 40

60 0

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60 0

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1000

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ing glucose, arabinose, xylose, galactose, rhamnose, and mannose residues in the cell wall (Ishii & Nishijima, 1995; Shibuya, 1984); however, there is little information about in-soluble bound phenolic acids in barley cultivated in Japan.

Table 2 shows the content of bound phenolic acids in pearled barley cultivated in Japan. Ferulic acid was the most abundant, followed by sinapic acid and caffeic acid. Unex-pectedly, p-coumaric acid was not detected, indicating only a small amount of p-coumaric acid below the limit of detection is present in these cultivars. Nordkvist et al. (1984) examined the distribution of insoluble bound phenolic acids in barley grains by gas chromatography and reported p-coumaric acids exist in their husk and aleurone layer with only trace amounts in the endosperm. As pearled barley samples (abraded 40% of dry matter) were used in this study, the samples were comprised of mostly endosperm. The content of ferulic acid obtained from the 20 barley samples ranged from 4.3 to 34.2

derivatives and the integrated peak area, various amounts of standard solution were analyzed. Good linearity according to Beer’s Law was obtained for concentrations of 0.02 to 0.08 μM for caffeic acid, 0.2 to 1.0 mM for p-coumaric acid, 20 to 100 μM for ferulic acid and 0.2 to 1.0 μM for sinapic acid; their linear regressions between peak area (y) and hydroxy-cinnamic acid content (x) were expressed as y = 10026x + 61993 (R=0.998) for caffeic acid, y = 6450357x + 575233 (R=0.996) for p-coumaric acid, y = 1448677x + 110515 (R=0.998) for ferulic acid, and y = 3177243x + 29918 (R=1.0) for sinapic acid. These results indicate high sensitivity with this HPLC method.

Insoluble bound phenolic acids Two hydroxycinnamic acid derivatives, ferulic acid and p-coumaric acid, are the major phenolic compounds in cereal and exist both in soluble free and insoluble bound forms (Sosulski et al., 1982). Most insoluble derivatives are bound to polysaccharides contain-

Table 2. Insoluble bound phenolic acids contained in extracts from the pearled barley samples.

Sample name

Insoluble bound phenolic acids

Caffeic acid(mg/100 g dry matter)

Ferulic acid(mg/100 g dry matter)

Sinapic acid(mg/100 g dry matter)

Pirkka 0.002 ± 0.000 7.4 ± 0.2 0.025 ± 0.005Kanto kawa 87 0.002 ± 0.001 7.1 ± 0.7 0.118 ± 0.036Kanto kawa 88 0.003 ± 0.000 9.8 ± 2.6 0.090 ± 0.024Touzan kawa 107 0.002 ± 0.000 6.5 ± 0.6 0.082 ± 0.010Kanto nijo 40 0.002 ± 0.001 7.5 ± 0.4 0.057 ± 0.038Kanto nijo 41 0.006 ± 0.001 6.5 ± 4.5 0.124 ± 0.029Kankei n551 0.006 ± 0.001 21.9 ± 2.9 0.445 ± 0.173Kankei n552 0.015 ± 0.005 30.7 ± 11.1 0.364 ± 0.084Kankei n553 0.010 ± 0.001 34.2 ± 8.4 0.399 ± 0.061Kankei n554 0.010 ± 0.001 30.1 ± 12.3 0.139 ± 0.061Daikei HQ10 0.004 ± 0.001 7.6 ± 2.1 0.083 ± 0.000BF 99-1 0.016 ± 0.004 12.9 ± 0.9 0.170 ± 0.018Daikei HL9-2-6 0.004 ± 0.000 11.8 ± 1.5 0.066 ± 0.025R 2815 0.002 ± 0.000 12.1 ± 2.9 0.085 ± 0.006Elrose 0.003 ± 0.000 8.9 ± 1.1 0.063 ± 0.003Stirling 0.002 ± 0.000 5.1 ± 1.3 0.029 ± 0.003M 737 0.011 ± 0.002 15.3 ± 1.0 0.230 ± 0.013Sukai golden 0.002 ± 0.000 4.3 ± 0.5 0.070 ± 0.019Scarlett 0.002 ± 0.000 5.3 ± 1.0 0.046 ± 0.005RISO 1508 0.007 ± 0.000 34.2 ± 4.8 tr

tr: detected but too small to quantify

Antioxidant Activities and Phenolic Acids of Barley 219

(Zielinski & Kozlowska, 2000; Liyana-Parthiraka & Shahidi, 2006). In this study, we determined the content of compounds containing catechin using HPLC. A sharp peak observed at 9.8 min corresponded to catechin (data not shown). We also determined the content of procyanidins as free phenolic com-pounds using spectrophotometry (vanillin assay).

As shown in Table 3, the content of procyanidins in the 20 barley samples ranged from 12.2 to 80.3 mg/100 g. Kankei n552 barley had the highest procyanidins content (80.3 mg/100 g) while Kanto kawa 88 barley had the lowest (12.2 mg/100 g). In contrast, Kankei n553 had the highest content of catechin (28.2 mg/100 g); no catechin was detect-ed in Kanto nijo 41 barley.

The total polyphenol content of the 20 barley samples ranged from 152.4 to 324.0 mg-GAE/100 g. Kankei n551 barley had the highest content of total polyphenols, while Scarlett barley had the lowest. The total polyphenol content

mg/100 g dry matter. Kankei n553 and RISO 1508 barley (produced in Tsukuba, Japan and Sweden) had the highest content of ferulic acid, while Sukai golden barley had the lowest. Kankei n551 also had the highest content of sinapic acid (0.445 mg/100 g), although no sinapic acid was detected in RISO 1508 barley. This is the first study to detect bound sinapic acid in barely.

The content of caffeic acid ranged from 0.002 to 0.016 mg/100 g; BF 99-1 showed the highest content. Four Kankei breeding lines (n551, n552, n553 and n554) contained a large amount of ferulic acid and sinapic acid, while three barley cultivars, Elrose (Canada), Stirling (Australia) and Sukai golden (Tochigi, Japan) contained only a small amount of fe-rulic acid, caffeic acid and sinapic acid.

Soluble free phenolic compounds, and total polyphenol content Phenolic compounds are a major group of com-pounds that contributed to the antioxidant activity of cereals

Table 3. Total polyphenol content and soluble free phenolic compounds contained in extracts from pearled barley samples.

Sample name Total polyphenol contents

(mg-GAE/100 g dry matter)

Soluble free phenolic compounds

Procyanidins(mg/100 g dry matter)

Catechin(mg/100 g dry matter)

Pirkka 184.5 ± 16.6 39.1 ± 3.2 9.8 ± 0.1Kanto kawa 87 175.8 ± 5.4 24.9 ± 4.5 9.8 ± 0.3Kanto kawa 88 165.4 ± 6.4 12.2 ± 0.8 0.1 ± 0.0Touzan kawa 107 159.3 ± 3.5 35.3 ± 0.8 3.2 ± 0.2Kanto nijo 40 202.0 ± 1.0 13.7 ± 4.5 9.7 ± 0.2Kanto nijo 41 157.1 ± 6.0 13.3 ± 4.4 trKankei n551 324.0 ± 6.2 50.9 ± 5.1 26.0 ± 0.2Kankei n552 284.3 ± 1.4 80.3 ± 21.8 7.9 ± 2.1Kankei n553 283.8 ± 4.4 53.0 ± 2.7 28.2 ± 1.6Kankei n554 261.5 ± 2.0 70.9 ± 10.2 10.7 ± 0.6Daikei HQ10 220.4 ± 7.7 33.6 ± 2.7 10.0 ± 1.3BF 99-1 294.6 ± 11.9 29.6 ± 0.6 3.9 ± 1.2Daikei HL9-2-6 197.3 ± 7.7 42.3 ± 14.9 5.3 ± 0.4R 2815 206.0 ± 7.6 27.4 ± 0.3 3.9 ± 0.3Elrose 153.4 ± 2.1 23.7 ± 1.3 9.0 ± 0.3Stirling 164.8 ± 4.1 48.3 ± 3.9 4.4 ± 0.1M 737 189.8 ± 1.9 51.5 ± 6.8 7.6 ± 1.1Sukai golden 185.7 ± 3.4 45.1 ± 1.6 7.7 ± 4.8Scarlett 152.4 ± 1.2 23.8 ± 0.8 3.3 ± 0.2RISO 1508 219.9 ± 4.0 56.0 ± 3.0 14.6 ± 1.1

GAE: gallic acid equivalents; tr: detected but too small to quantify

a.�YoShida�et al.220

activity of barley. This finding was observed for14 malting barley varieties, which differed significantly in their antioxi-dant properties (Zhao et al., 2008). In the cultivars showing no activity (Kanto kawa 88 and Kanto nijo 41), only a small amount of total polyphenols (165.4 mg-GAE/100 g and 157.1 mg-GAE/100 g, respectively; Table 3) was detected.

ORAC ORAC of barley samples (except for Kanto kawa 88 and Kanto nijo 41) ranged from 1050 to 3816 μmol-TE/100 g. Kankei n551 had the highest ORAC while Scarlett had the lowest (Table 4).

Correlation between antioxidant activity, total polyphenol content, and contents of soluble free and insoluble bound phenolic acids Phenolic compounds have been reported to be responsible for the antioxidant activity of vegetables and cereals (Millard et al., 1996; Oki et al., 2002; Beta et al., 2005). To clarify the relationship between antioxidant activity and contents of phenolic compounds in barley, the correla-tion between antioxidant activity (DPPH and ORAC assays)

was relative to the content of procyanidins and catechin.DPPH radical scavenging activity DPPH is a relatively

stable organic radical. It has been widely used for the deter-mination of antioxidant activity of antioxidant compounds as well as various cereal extracts (Goupy et al., 1999; Zhao et al., 2006). To evaluate antioxidant activity of barley, DPPH radical scavenging activity of 20 samples was measured and compared (Table 4). Of the 20 barley samples (except for Kanto kawa 88 and Kanto nijo 41), 18 exhibited DPPH radical scavenging activity at the tested concentration; the activity of these 18 barley samples ranged from 403 to 1501 μmol-TE/100 g. Kankei n551 has the strongest DPPH radi-cal scavenging activity among all the samples, while Kanto kawa 87 barley had the lowest. The three Kankei breeding lines (n552, n553 and n554) also showed high DPPH radi-cal scavenging activity. The significant differences in DPPH radical scavenging activity among barley cultivars suggest that the cultivar has a significant influence on the antioxidant

Table 4. DPPH radical scavenging activity and ORAC in extracts from pearled barley samples.

Sample name DPPH

(µmol-TE/100 g dry matter)ORAC

(µmol-TE/100 g dry matter)

Pirkka 687 ± 11 1500 ± 141Kanto kawa 87 403 ± 78 1300 ± 0Kanto kawa 88 tr trTouzan kawa 107 420 ± 3 1100 ± 0Kanto nijo 40 600 ± 71 1400 ± 141Kanto nijo 41 tr trKankei n551 1501 ± 223 3816 ± 22Kankei n552 1406 ± 140 3161 ± 55Kankei n553 1308 ± 12 3194 ± 132Kankei n554 1115 ± 29 2769 ± 98Daikei HQ10 731 ± 38 1719 ± 115BF 99-1 694 ± 25 1910 ± 297Daikei HL9-2-6 551 ± 4 1400 ± 141R 2815 671 ± 7 1700 ± 0Elrose 509 ± 37 1250 ± 71Stirling 479 ± 26 1500 ± 0M 737 745 ± 11 2050 ± 71Sukai golden 495 ± 5 1200 ± 141Scarlett 428 ± 2 1050 ± 71RISO 1508 893 ± 11 2500 ± 141

DPPH: 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, ORAC: oxygen radical absorbance capacity, TE: Trolox equivalents, tr: detected but too small to quantify

Antioxidant Activities and Phenolic Acids of Barley 221

In general, the various methods used to determine the an-tioxidant activity are based on different reaction mechanisms, thus giving varying results. Therefore, we analyzed both the DPPH radical scavenging activity and ORAC to evaluate an-tioxidant activity in barley. To correlate the results obtained by both methods, a regression analysis was performed. Both DPPH radical scavenging activity and ORAC showed good, positive correlation (0.980; p<0.01).

In conclusion, we developed an analytical method using alkaline hydrolysis and HPLC-ECD to determine the con-tent of insoluble bound phenolic acids such as caffeic acid, p-coumaric acid, ferulic acid and sinapic acid, in pearled bar-ley. The present results confirm the usefulness of detection and quantification of insoluble bound phenolic acids using this method. Insoluble bound phenolic acid content in five pearled cultivars and 15 breeding lines of barley was: ferulic acid (4.3-34.2 mg/100 g); sinapic acid (0.025-0.445 mg/100 g); caffeic acid (0.002-0.016 mg/100 g); soluble free poly-phenol content was: procyanidins (12.2-80.3 mg/100 g) and catechin (0.1-28.2 mg/100 g); and total polyphenol content was: 152.4-324.0 mg-GAE/100 g.

To evaluate the antioxidants of 20 barley samples, DPPH radical scavenging activity and ORAC were measured. A high correlation coefficient (0.980, p<0.01) between the two methods was found. Total polyphenol content was positively correlated with DPPH racical scavenging activity (0.875, p<0.01) and ORAC (0.881, p<0.01). The correlation be-tween bound phenolic acid and total polyphenol contents was

and contents of total polyphenols, soluble free and insoluble bound phenolic acids were investigated. The results are pre-sented in Table 5.

The highest correlation was found between the ORAC and bound sinapic acids (0.917, p<0.01), and the lowest cor-relation was found between DPPH radical scavenging activ-ity and bound caffeic acids (0.616, p<0.01). Total polyphenol content had a high correlation with DPPH radical scavenging activity (0.875, p<0.01) and ORAC (0.881, p<0.01) as re-ported previously (Zhao et al., 2008). Surprisingly, insoluble bound phenolic acid content showed a positive high cor-relation with DPPH radical scavenging activity and ORAC (0.616-0.917). Bound ferulic acid content (abundant phenolic acids) showed a higher correlation with DPPH radical scav-enging activity (0.837, p<0.01) and ORAC (0.857, p<0.01) than free polyphenol content. Liyana-Pathiraka and Shahidi (2006) reported that the content of insoluble bound phenolic compounds was significantlly higher than that of soluble free phenolic compounds, and that bound phenolic compounds demonstrated a significantly higher antioxidant capacity than that of free phenolic compounds in wheat. Similarly, in-soluble bound phenolic acids also contributed to antioxidant activity in barley. The correlation between the contents of insoluble bound phenolic acids and total polyphenols (0.695-0.797, p<0.01) was relatively higher than that between the contents of soluble free polyphenols and total polyphenols (0.595-0.627, p<0.01).

Table 5. Correlation coefficients between antioxidant, free polyphenols and bound phenolic acids.

Antioxidant activity Free polyphenols Bound phenolic acids

DPPH ORAC TPC PRO CAT FER CAF SIN

DPPH 1 0.980** 0.875** 0.732** 0.756** 0.837** 0.616** 0.890**

ORAC — 1 0.881** 0.726** 0.750** 0.857** 0.630** 0.917**

TPC — — 1 0.595** 0.627** 0.714** 0.695** 0.797**

PRO — — — 1 0.407 0.738** 0.549* 0.552*

CAT — — — — 1 0.611** 0.203 0.738**

FER — — — — — 1 0.625** 0.805**

CAF — — — — — — 1 0.643**

SIN — — — — — — — 1

Pearson correlation analysis was performed using SPSS software to determine the correlations among the means of anti-oxidant activities, total polyphenol contents and free and bound phenolic acids.TPC: total phenolic contents; PRO: procyanidins; CAT: catechin; FER: ferulic acid; CAF: caffeic acid; SIN: sinapic acid.* p< 0.05, ** p< 0.01.

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