Variations in vitamin E, phenolic content, and antioxidant properties of different wheat cultivars of South Korea

ABSTRACT The vitamin E, total phenolic content (TPC), and antioxidant activity (AA) profiles of 32 whole wheat cultivars were investigated. Samples were classified based on its hardness (HW: hard wheat, MW: medium wheat, and SW: soft wheat) for easy identification. Total vitamin E content ranged from 64.38SW2 to 111.83MW9 mg/kg; α-tocopherol (6.03MW13 to 21.98MW2 mg/kg), β-T (2.14MW8 to 21.01MW9 mg/kg), α-tocotrienol (4.28MW12 to 9.34MW9 mg/kg), and β-T3 (38.71MW16 to 61.07MW9 mg/kg) were the predominant homologs, with β-T3 as the highest. AA ranged from 334.40MW4 to 584.80HW4 μmol gallic acid equivalent/kg while TPC varied from 1139.70SW6 to 2772.60MW1 μmol GAE/kg. Significant differences in the vitamin E content, TPC, and AA were found among the cultivars regardless of its hardness index. A very weak correlation was found between TPC and AA while a moderate negative correlation was observed between vitamin E content and AA. Results from this study can serve as basis of further studies on the bioactive profiles of Korean wheat cultivars.


Introduction
Whole grains are cereal grains whose endosperm, germ, and bran are intact prior to cracking or flaking. Most of the nutritive and bioactive components of grains are concentrated in its bran and germ, which are removed during milling. Several researchers have proved that there is a strong relationship between the frequent whole grain diet and body defense mechanism against oxidative stress, which is related to chronic diseases such as hypertension, diabetes, and stroke (Liu, 2007;Lobo, Patil, Phatak, & Chandra, 2010;Tabassum & Ahmad, 2011). These health benefits of whole grains have been known to be primarily due to the presence of phytochemicals. Phytochemicals are natural non-nutritive bioactive components in plant having radical scavenging property. Such components present in grains are said to be affected by cultivars (Tabassum & Ahmad, 2011). Thus, prior information on bioactive compounds of different wheat cultivars is highly needed for further processing and storage; however, information on whole wheat cultivars developed and grown in Korea is limited.
In Korea, busy lifestyle contributed to the increased consumption of wheat-based fast food diet such as bread instead of longtime staple whole grain rice. Majority of wheat-based products are consumed from refined wheat rather than whole grain. In this study, inherent differences in the contents of vitamin E, total phenolic content (TPC), and antioxidant activity (AA) of 32 South Korean wheat cultivars were investigated in order to provide basic information which could encourage the consumption of whole wheat grains.

Sample and sample preparation
A total of 32 Korean wheat cultivars developed in the National Institute of Crop Science, Rural Development Administration, were cultivated in the experimental research field located in southern area (Iksan, Jeonbuk, Korea) in the 2015-2016 crop seasons. The seeds were planted in late October in 2015 and each plot consisted of three 4 m rows spaced 25 cm apart, and plots were combined-harvested in mid-June in 2016. The harvested wheat cultivars were classified into three categories based on hardnesshard, medium, and soft wheat (HW, MW and SW, respectively) for easy identification (Table 1). Whole wheat flour was prepared by using a hammer mill (Laboratory Mill 3100, Pertent Co. Ltd., Huddinge, Sweden) equipped with a 0.5 mm screen. Whole wheat flour was stored at −20°C prior to analysis.

Extraction of vitamin E
Vitamin E extraction was carried out according to the saponification method of Chun, Lee, Ye, Exler, and Eitenmiller (2006) with minor modifications. Ground sample (2 g) was mixed with 10 mL of pyrogallol in ethanol (6%, w/v) and 8 mL of potassium hydroxide in water (60%, w/v) and heated at 80°C for 1 h with shaking. Then, 20 mL of 2% sodium chloride in deionized water was added and extracted thrice with 15 mL solvent (hexane:ethyl acetate = 85:15, v/v) containing 0.01% butylated hydroxytoluene. A 2-mL aliquot was dried under nitrogen gas, re-dissolved in 1.0 mL n-hexane, and filtered through 0.45 μm membrane filter (PTFE, Advantec, Tokyo, Japan) prior to high performance liquid chromatography (HPLC) analysis.

HPLC conditions for vitamin E analysis
All vitamin E homologs (α-, β-, γ-, δ-tocopherols (T) and tocotrienols (T3)) of sample extracts were analyzed using an HPLC system (RF-10AXL, Shimadzu Co., Kyoto, Japan) equipped with fluorescence detector and a LiChrospher® Diol 100 column (4.6 mm ⅹ 250 mm, 5 μm) (Merck, Darmstadt, Germany). Injection volume used was 20 µL; isocratic mobile phase was 1% isopropanol in n-hexane at a flow rate of 1 mL/min. Vitamin E was detected at 285 nm for excitation and 325 nm for emission. Peaks were identified by comparison of retention times to the standards. Quantitation was done as described by Chun et al. (2006) and the vitamin E content was expressed as mg/kg flour.

Determination of TPC
TPC of whole wheat flour was determined according to the method of Singleton, Orthofer, and Lamuela-Raventós (1999) after minor modification. Briefly, to 0.5 g flour, 5 ml of 80% aqueous methanol was added and heated at 60°C for 2 h with shaking. The samples were centrifuged at 2000 rpm for 5 min. Exactly 200 μL of supernatant was mixed with 1000 μL of deionized water. Then, 100 μL Folin-Ciocalteu's phenol reagent (reagent:deionized water = 1:1, v/v) was added followed by 200 μL of 5% sodium carbonate in deionized water (w/v) and mixed thoroughly. The tubes were placed in the dark for 1 h; absorbance was determined at 750 nm using a spectrophotometer (Eon, Biotek Instruments, Seoul, Korea). All experiments were conducted in triplicate, and the TPC was expressed as μmol GAE/kg of whole wheat flour.

Determination of AA
AA was evaluated by diphenylpicrylhydrazyl (DPPH) radical scavenging assay based on a method described by Abdelhady, Motaal, and Beerhues (2011) with modifications. Test extracts prepared for TPC determination were also used for AA. Briefly, 200 μL of sample was mixed with 800 μL of 0.1 mM DPPH solution. The samples were kept in the dark for 30 min and then absorbance of the samples at 517 nm was measured using a spectrophotometer. All experiments were conducted in triplicate, and the AA was expressed as μmol GAE/kg of whole wheat flour.

Statistical analysis
Statistical analysis was done by means of SPSS statistical package (IBM Corp., Released 2013, IBM SPSS Statistics for Windows, Version 22.0, Armonk, NY, USA) using ANOVA and Duncan's multiple range test. A P < 0.05 was taken as statistically significant.

Vitamin E content of Korean whole wheat cultivars
Vitamin E is a well-known representative antioxidant in plants.
It is comprised of a total of eight homologs: α-T, β-T, γ-T, δ-T, α-T3, β-T3, γ-T3, and δ-T3. They have different biological activities depending on their structures. As shown in Table 2, β-T3, the total vitamin E content, ranged from 64.38 SW2 to 111.83 MW9 mg/kg. Regardless of its hardness (HW, MW, or SW), high and low values of T and T3 were seen among all samples. With regard to T, α-T (6.03 MW13 to 21.98 MW2 mg/kg) and β-T (2.14 MW8 to 21.01 MW9 mg/kg) were the predominant vitamin E homologs. For T3, on the other hand, β-T3 (38.71 MW16 to 61.07 MW9 mg/kg) was found in greater amount than α-T3 (4.28 MW12 to 9.34 MW9 mg/kg). Among the eight homologs, β-T3 was predominant in all of the 32 wheat cultivars, constituting about 60% of the total vitamin E content. In a study on vitamin E content of six wheat varieties in the United States, β-T3 was also found to be the predominant homolog (Okarter, Liu, Sorrells, & Liu, 2010). Also, in another study it was observed that αand β-T were predominant in the germ fraction of einkorn wheat while αand β-T3 levels were highest in its bran fraction (Hidalgo & Brandolini, 2008). In a study by Engelsen and Hansen (2009), the content of tocopherols and tocotrienols varied depending on the wheat fractions. The content of tocotrienols was distributed uniformly in the wheat grain with the highest content in the bran fractions. High levels of β-T3 reported in the present study may also be attributed to the presence of bran.
Furthermore, it was reported that large variations in vitamin E contents could be observed depending on wheat genotypes and phenotypes (Lampi, Nurmi, Ollilainen, & Piironen, 2008). As shown in Table 2, significant differences were found among the cultivars.

TPCs of Korean whole wheat cultivars
TPC of the 32 whole wheat cultivars ranged from 1139.70 SW6 to 2772.60 MW1 (Table 3). TPC of the wheat cultivars varied regardless of its hardness. Several previous studies reported various ranges of TPC depending on wheat varieties and cultivation regions: 113-371 μg of GAE/g for hard winter wheat (Yu, Haley, Perret, & Harris, 2004), 235-470 μmol GAE/100 g for Maryland-grown soft wheat (Moore et al., 2005), and 841-1099 μmol GAE/100 g for US-grown wheat (Okarter et al., 2010). In the present study, significant differences in TPC were also found among the cultivars.
In this study, phenolics were extracted using aqueous methanol. It is said that phenolic content could be affected by extraction methods. Zieliński and Kozłowska (2000) reported 292.12 μmol GAE/100 g TPC in cereal grains using 80% methanol as extraction solvent. Yu, Haley, Perret, and Harris (2002) used Soxhlet extraction; reported values ranged from 287 to 545.76 μmol GAE/100 g. Moreover, alkali hydrolysis procedure could be performed prior to organic solvent extraction method in order to extract insolublebound phenolics (Liyana-Pathirana & Shahidi, 2006). In this study, samples showed generally lower TPC values than those reported by previous studies. It might be due to the absence of an alkali hydrolysis prior to organic solvent extraction. Variations depending on extraction method need to be further investigated in the future.

AA of Korean whole wheat cultivars
DPPH radical scavenging assay was conducted to determine the AA of Korean wheat cultivars, and the results are shown in Table 3. AA ranged from 334.40 MW4 to 584.80 HW4 . Significant differences in AA were found among the cultivars; high and low values of AA can be seen among the flour samples regardless of its hardness. Antioxidant compounds prevent free radical-induced oxidative stress and provide health benefits when consumed. AA could be affected by the number of hydroxyl/amino groups and the position of chemical components within molecules (Cai, Sun, & Corke, 2003). In this study, AA was evaluated using DPPH assay. Many published studies are available with regard to AA of wheat using DPPH assay. However, because various extraction methods and standards were employed, it was difficult to compare the results of the present study with those of previous related studies. Nevertheless, AA was observed in all of the wheat cultivars. The results indicate that these were good sources of bioactive compounds.

Relationship between TPC, vitamin E content, and AA
The correlation coefficient (R) between TPC and AA and between vitamin E content and AA was also determined.
The computed values were 0.1992 and −0.4058, respectively. A very weak correlation was seen between TPC and AA while a moderate negative correlation was observed between TPC and vitamin E content. It was reported that although many studies on different food samples show that AA is highly correlated with the components commonly associated with the antioxidant property of a food, it is not applicable for all due to the presence of other food components such as pigments, ascorbates, and others (Babbar, Oberoi, Uppal, & Patil, 2011;Li, Yuan, Yang, Tao, & Ming, 2013).

Conclusions
This study was conducted to investigate variations in vitamin E profile, TPC, and AA of wheat cultivars grown in South Korea. The β-T3 was the predominant vitamin E homolog which comprised 60% of total vitamin E content. Significant differences in TPC, AA, and vitamin E content were observed among the analyzed wheat cultivars. Results from this study can serve as basis of further bioactive profiling and storage studies of wheat cultivars in Korea, consequently encouraging the consumption of whole wheat grains.

Disclosure statement
No potential conflict of interest was reported by the authors.