J. Agric. Food Chem. 2010, 58, 9235–9241
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DOI:10.1021/jf101700s
Comparison of Antioxidant Activities of Different Colored Wheat Grains and Analysis of Phenolic Compounds QIN LIU,†,‡ YANG QIU,† AND TRUST BETA*,†,§ †
Department of Food Science, University of Manitoba, Winnipeg, Manitoba, Canada R3T 2N2, School of Food Science and Engineering, Nanjing University of Finance and Economics, Jiangsu Key Laboratory of Quality Control and Processing for Cereals and Oils, Nanjing, Jiangsu, People’s Republic of China 210003, and §Richardson Centre for Functional Foods and Nutraceuticals, Smartpark, University of Manitoba, Winnipeg, Manitoba, Canada R3T 6C4 ‡
Extracts from six wheat varieties (three purple, one yellow, two red, and one white) were evaluated and compared for their antioxidant capacities against oxygen radical and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. Phenolic composition in the extracts was examined by high-performance liquid chromatography and mass spectrometry. The results showed that Charcoal purple wheat had remarkable antioxidant activity (up to 6899 μmol/100 g) followed by Red Fife wheat and yellow Luteus wheat. White AC Vista wheat, due to its lowest phenolic content, exhibited the weakest antioxidant property. The major phenolic composition identified in wheat grains consisted of phenolic acids, flavones, flavonols, and anthocyanins. The former three components were detected in all of the wheat varieties, whereas anthocyanins were identified only in purple wheat. Therefore, anthocyanins could be the major compounds distinguishing purple wheats from other colored wheats with high antioxidant activity. KEYWORDS: Colored wheat grain; antioxidant activity; phenolic acid; flavonoid; anthocyanin
INTRODUCTION
Wheat is a crop that is cultivated worldwide and consumed as a staple food in many countries. On the market, most wheats are white- or red- grained. Some novel wheat varieties such as purple and blue wheat grains are also commercially available, but in limited amounts. As an important food ingredient, wheat grain is usually ground into flour to make bread, cake, pasta, noodle, or other food products. In recent years, whole grain food products are more favorable than those made from refined grain flour because they have been reported to lower the incidence of chronic diseases such as type II diabetes, cardiovascular diseases, and some cancers (1). These health benefits have been partly attributed to the unique phytochemicals present in whole grain (1). Therefore, the full characterization of these health-benefiting substances in wheat grain is important for the agricultural industry in breeding and developing value-added wheat containing enhanced nutritional and health-promoting compounds. Phenolic compounds are among phytochemicals in cereal grains that are actively being investigated as natural antioxidants. The major phenolics reported in wheat are phenolic acids and flavonoids. Although these compounds exist at low concentration, they substantially influence the quality of wheat. The antioxidant activities of different wheat grains and their phenolic profiles have been investigated by several research groups (2-4). However, most of them are focused on white or red wheat varieties. To our knowledge, there is no report to compare the antioxidant properties of purple, red, yellow, and white wheat
grains. Furthermore, the inherent varietal differences in phenolic compounds between different colored wheats have not yet been reported. Thus, the objectives of this study were to compare the antioxidant activities of purple, red, yellow, and white wheat grains and to qualitatively and quantitatively analyze phenolic acids and flavonoids in different colored wheat grains. MATERIALS AND METHODS
*Corresponding author [phone (204) 474-8214, (204) 474-6612; fax (204) 474-7630; e-mail
[email protected]].
Chemicals and Standards. Analytical grade acetic acid used in the extraction and Folin-Ciocalteu reagent were purchased from Fisher Scientific (Pittsburgh, PA). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), fluorescein, 2,20 -azobis(2-amidinopropane) dihydrochloride (AAPH), 6-hydroxy2,5,7,8-tetramethylchroman-2- carboxylic acid (Trolox), catechin, and phenolic acid standards were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). HPLC grade acetone and methanol were used in the extraction. MS grade water, acetonitrile, and acetic acid were used in LCMS analysis. All of the HPLC grade and MS grade solvents were purchased from Sigma-Aldrich Chemical Co. Sample Description and Preparation. Six different colored bread wheat (Triticum aestivum) grains were used in this study including three purple wheats (Charcoal, Indigo, and Konini), one red wheat (Red Fife), one yellow wheat (Luteus), and one white wheat (AC Vista). The wheat varieties were grown under the same organic conditions in 2008. Among them, the three purple wheats, Charcoal, Indigo, and Konini, were grown in the same location at the Danish Agricultural Museum, Gl. Estrup, in Jutland, Denmark (latitute N 56° 26.2960 , longitude E 010° 20.5220 ). The other three wheats were grown at Mørdrupgard, a farm in Lynge north of Copenhagen, Denmark (latitude N 55° 49.5300 , longitude E 012° 13.5870 ). Before analysis, whole grain samples were ground into a fine powder by using an ultracentrifugal mill (Retsch Inc., Haan, Germany) equipped with an 80 mesh sieve (0.5 mm) and stored at -20 °C before extraction. The moisture contents of whole wheat
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flour were determined by AOAC (2003) official method 925.10 (5) before extraction. Extraction of Soluble Phenolic Compounds. The soluble phenolic compounds were extracted with two solvent systems. Methanol/1 M HCl (85:15, v/v) was used to extract anthocyanins because anthocyanins are more stable and extractable when the pH 81.38 mg/100 g). Red and yellow wheat grains contained significantly higher concentrations of p-coumaric acid than other wheats. White wheat, although containing comparable vanillic acid, caffeic acid, and p-coumaric acid as red and yellow
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Liu et al. a
Table 1. Contents of Identified Phenolic Acids in Tested Purple, Red, Yellow, and White Wheat Grains
monomeric phenolic acid (mg/100 g) variety
vanillic
caffeic
p-coumaric
ferulic
sinapic
Charcoal Indigo Konini Red Fife Luteus AC Vista
3.35 ( 0.74ab 3.21 ( 0.65ab 2.58 ( 0.64bc 1.34 ( 0.60d 1.34 ( 0.62d 1.32 ( 0.22d
0.95 ( 0.07bc 0.84 ( 0.15dc 1.40 ( 0.14a 0.99 ( 0.22bc 1.07 ( 0.17abc 0.83 ( 0.17c
2.87 ( 0.12a 2.12 ( 0.20a 2.38 ( 0.26a 3.56 ( 0.32a 3.31 ( 0.51e 2.73 ( 0.41a
87.37 ( 1.42a 86.59 ( 0.42a 81.38 ( 1.70b 69.31 ( 0.04d 80.98 ( 1.34b 74.35 ( 0.14c
3.04 ( 0.08a 2.36 ( 0.11b 1.93 ( 0.21c 2.40 ( 0.17b 1.39 ( 0.09d 0.70 ( 0.23e
diferulic acid (mg/100 g) 0
variety
5-5
Charcoal Indigo Konini Red Fife Luteus AC Vista
1.13 ( 0.15b 0.89 ( 0.18 cd 1.62 ( 0.26ab 0.80 ( 0.15d 0.92 ( 0.05c 0.88 ( 0.10d
a
8-O-40
8-50
6.62 ( 0.61a 5.31 ( 0.99b 6.57 ( 0.81a 5.08 ( 0.62b 4.70 ( 0.99c 3.26 ( 1.06d
9.09 ( 0.44b 8.26 ( 0.25c 11.7 ( 0.31a 8.87 ( 1.26c 12.7 ( 0.96a 9.73 ( 0.47b
The response factors of 5-50 , 8-O-40 , and 8-50 diferulic acids are 0.21, 0.14, and 0.12, respectively. Means in the same column with different letters are significantly different.
Table 2. Total Flavonoid Content and Total Anthocyanidin Content in Different Colored Wheat Grainsa grain color
variety
total flavonoid content
total anthocyanidin content (color of extract)
purple
Charcoal Indigo Konini Red Fife Luteus AC Vista
102.95 ( 1.15a 35.79 ( 0.25b 21.59 ( 0.58c 10.72 ( 0.84de 13.44 ( 0.76d 9.60 ( 0.43e
23.45 ( 0.06a (purple-red) 7.24 ( 0.04b (red) 2.54 ( 0.04c (pink) 0.96 ( 0.06d (yellow) 0.95 ( 0.03d (yellow) 0.77 ( 0.03d (yellow)
red yellow white
a TFC and TAC are, respectively, expressed as mg of catechin equivalents and cyanidin-3-glucoside equivalents/100 g of wheat grain (dry weight basis). Means in the same column with different letters are significantly different.
wheat grains, had a significantly lower level of sinapic acid. Hence, these substantial differences of phenolic acid content between white wheat and other colored wheat grains can mainly be attributed to ferulic acid and sinapic acid. Besides the above five monomeric phenolic acids, three phenolic acid dehydrodimers were also detected in this study. Ferulate dehydrodimers also referred to as diferulic acids, are the most common dimeric phenolic acids observed in cereal grains. They are abundant in the cell wall and occur only in the insoluble fraction (16). By plotting the typical molecular ions at m/z 385 expected from diferulic acids, three peaks were observed with retention times at 52.15, 53.07, and 63.40 min, respectively, on the reversed-phrase HPLC. By comparing the experimental data with the literature (8, 17, 18), these peaks were respectively assigned as 5-50 , 8-O-40 , and 8-50 (benzofuran form) diferulic acids. The quantitative analysis of each ferulic acid dehydrodimer was accomplished by using response factors at 280 nm against transcinnamic acid (18). As seen in Table 1, 8-50 (benzofuran form) diferulic acids was found as the most abundant ferulic acid dehydrodimer, ranging from 8.3 to 12.7 mg/100 g. The amounts of 8-O-40 and 5-50 diferulic acids in wheat grains varied from 3.3 to 6.6 mg/100 g and from 0.8 to 1.6 mg/100 g, respectively. They were more concentrated in purple wheat. White wheat had a significantly lower level of 8-O-40 diferulic acids than other colored wheats. By calculation of the total amount of phenolic acids in each wheat grain, it can be seen that white wheat contained the least phenolic acids and the lowest total phenolic content. Flavonoid Composition. Flavonoids constitute a large group of phenolic compounds in plants, accounting for two-thirds of the
dietary polyphenols (19). The total flavonoid contents in purple, red, yellow, and white wheat are presented in Table 2 and expressed as milligrams of catechin equivalents per 100 g of wheat grain. Among all of the colored wheat grains, purple wheat was found to possess significantly highest flavonoid content, ranging from 21.59 to 102.95 mg/100 g. Moderate flavonoid contents were found in yellow and red wheats, with levels of 13.44 and 10.72 mg/100 g, respectively. White wheat had the lowest total flavonoid content (9.6 mg/100 g). These results indicate that dark-colored wheat comprises more flavonoid content than lightcolored wheat. Anthocyanins are water-soluble pigments contributing to the blue, purple, and red color of cereal grains. Because of their antioxidant properties, anthocyanins are among the major flavonoids studied in cereal grains. The total anthocyanin contents in different colored wheat grains were determined in their acidic methanol extracts and expressed as milligrams of cyanidin-3glucoside equivalents per 100 g of wheat grain. As seen in Table 2, significant differences in anthocyanin contents were detected between purple and other light-colored wheats. Three purple wheats (Charcoal, Indigo, and Konini) were found to contain remarkable anthocyanin contents ranging from 2.5 to 23.5 mg/ 100 g. Their extracts, respectively, exhibited purple-red, red, and pink colors. However, the extracts from red, yellow, and white wheat grains appeared in a light yellow color. The total anthocyanin contents of these light-colored wheats were found to be
