Research Article Received: 30 September 2013
Revised: 26 September 2014
Accepted article published: 17 October 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6963
A correlation between tocopherol content and antioxidant activity in seeds and germinating seeds of soybean cultivars Yu Young Lee,a† Hyang Mi Park,a† Tae Young Hwang,a Sun Lim Kim,a Mi Jung Kim,a Seuk Ki Lee,a Min Jung Seo,a Kee Jong Kim,a Young-Up Kwon,a Sang Chul Leeb and Yul Ho Kima* Abstract BACKGROUND: Tocopherols are crucial lipid-soluble antioxidants and essential nutrients. There is increasing interest in the biofortification of crops with vitamin E for reducing micronutrient malnutrition. However, relatively little is known about the development of soybean cultivars with high levels of tocopherol through combined breeding. RESULT: Tocopherol contents of seeds and germinating seeds of 28 Korean soybean cultivars were analyzed and evaluated for health-promoting activities. Total tocopherol concentrations ranged from 203.9 to 503.1 𝛍g g−1 in seeds and from 20.1 to 230.1 𝛍g g−1 in germinating seeds. The traditional landraces of HaNagari (HN, 503.1 𝛍g g−1 ), Orialtae (OL, 486.6 𝛍g g−1 ), SuMoktae (SM, 476.5 𝛍g g−1 ) and SoRitae (SR, 475.5 𝛍g g−1 ) showed high levels of tocopherol content. The contents of the four isomers of tocopherol in seeds and germinating seeds were correlated with lipid peroxidation. The 𝜸- and 𝜹-tocopherol contents in seeds were related to 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activity (0.434; P < 0.01 and 0.373; P < 0.05). CONCLUSION: Total tocopherol content was higher in soybean landraces as compared with modern cultivars developed by cross-breeding. These results suggest that soybean breeding is necessary to increase tocopherol levels. © 2014 Society of Chemical Industry Keywords: antioxidant activity; lipid peroxidation; seed; germination; soybean; tocopherols
INTRODUCTION Tocopherols, generally known as vitamin E, are crucial lipid-soluble antioxidants. Vitamin E consists of a polar chromanol ring linked to a 15-carbon tail and comprises eight chemically distinct compounds that are separated into tocopherols and tocotrienols according to hydrophobic tail saturation. Tocopherols contain a fully saturated tail, whereas tocotrienols have three unsaturated double bonds. The different tocopherol and tocotrienol isomers, i.e. 𝛼, 𝛽, 𝛾 and 𝛿, are distinguished by the locations of methyl groups on the chromanol ring.1,2 Tocopherols play a significant role in food preservation and disease prevention.3 It is commonly believed that the beneficial effects of vitamin E are derived from preventing the breakdown of polyunsaturated fatty acids in membrane lipids and influencing cellular responses to oxidative stress in signal transduction pathways.4 Soybean (Glycine max L.) is an important highland crop consumed in various forms such as soybean oil, soybean sprout, soy paste, soymilk and tofu.5,6 Soybean oil corresponds to 20–30% of the vegetable oils world market and is an important source of natural tocopherols.7,8 Soybean sprouts, in particular, have been consumed as a year-round vegetable for thousands of years in Korea. There are excellent sources of nutrients and vitamins.9 Annually, more than 500 000 tons of soybean sprouts are consumed as vegetable in soups, salads and side dishes in Korea.10 At present, the J Sci Food Agric (2014)
market value for soybean sprouts is around $700 million and the consumption levels have been greatly increased by the diversification of cooking methods and the creation of new demand.11,12 However, relatively little is known about the impact of soybean germination on tocopherols. Many published papers have investigated tocopherol concentrations in soybean. The range and mean contents of four tocopherol isomers in seeds of five cultivars (DaewonKong, Taekwangkong, Jinpumkong, Hwaseongputkong and Ilpumgumjeongkong) was reported to be 305–389 μg g−1 dry weight.13 Five cultivars of black soybeans contained 249–405 μg g−1 of 𝛾-tocopherol that underwent significant degradation within a month of storage at room temperature (P < 0.01) but remained stable for 6 months at 4 ∘ C
∗
Correspondence to: Yul Ho Kim, National Institute of Crop Science, Rural Development Administration, Suwon 441-857, Republic of Korea. E-mail: [email protected]
† These authors contributed equally to this work. a National Institute of Crop Science, Rural Development Administration, Suwon, 441-857, Republic of Korea b School of Applied Bioscience, College of Agriculture and Life Sciences, Kyungpook National University, Daegu, 702-701, Republic of Korea
www.soci.org
© 2014 Society of Chemical Industry
www.soci.org and 14 months at −80 ∘ C.14 The tocopherol contents of soybean germplasms originating from different countries (USA, China and Korea) were also investigated in addition to seed size on the basis of 100-seed weight. Among Chinese soybeans, 13–24g of 100-seed weight group had the highest (256.1 μg g−1 ) average total tocopherol content.15 In the last decade, a complementary approach for development of micronutrient-dense crops. termed biofortification, has been undertaken with the aim of reducing micronutrient malnutrition and preventing diseases using genetic engineering approaches. Significant progress has been made in the development of crops biofortified with vitamin E and altered tocopherol or tocotrienol compositions.16 – 18 To date, no attempts have been made to develop a soybean cultivar with a high level of vitamin E using combined breeding in Korea. In this study, the concentrations of tocopherols in seeds and germinating seeds of 28 soybean cultivars were determined to find a tocopherol-rich soybean for biofortification. In addition, the relationship between the concentration of tocopherol isomers and antioxidant activities using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′ -azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) assay and lipid peroxidation inhibition test was investigated. The association between vitamin E isomer content and antioxidant activity was investigated in soybean seeds and germinating seeds.
YY Lee et al.
MATERIALS AND METHODS Chemicals and Instruments Ethanol, butylated hydroxytoluene (BHT), DPPH, ABTS, potassium persulfate, trichloroacetic acid and tocopherols standards were obtained from Sigma Chemical Co. (St Louis, MO, USA). Ethyl acetate (EtOAc), n-hexane, isopropanol and water were purchased from JT Baker (Phillipsburg, NJ, USA). All solvents and reagents used were analytical grade. Thiobarbituric acid (TBA) was supplied by MP Biomedicals, while tocotrienols were purchased from Davos Life Science (Tuas, Singapore). The tocopherol and tocotrienol contents of soybeans were determined by normal-phase high-performance liquid chromatography (HPLC). The HPLC system consisted of a solvent delivery pump (model 515; Waters, Milford, MA, USA) equipped with a spectrofluorometric detector (model 2475, Waters) and a Lichrosorb Si60 column (4.6 × 250 mm, 5 μm; Hibar, Darmstadt, Germany). The isocratic mobile phase was n-hexane:isopropanol (99:1, v/v) at a flow rate of 1.5 mL min−1 . Excitation and emission spectra were 290 and 330 nm, respectively. The injection volume was 10 μL and the column temperature was regulated at 30 ∘ C. Samples Twenty-eight cultivars of soybean were used in the experiment (Table 1). All soybeans were harvested on 24 October 2011 at
Table 1. Characteristics of 28 soybean cultivars Cultivar
Registration year
SoRitae (SR) SuMoktae (SM) Orialtae (OL) HaNagari (HN) JangDanBaemok (JD) ChungBukbaek (CB) GwangDu (GD) GwangKyo (GK) BaegCheon (BC) JangYeopkong (JY) BaekUnkong (BU) PalDalkong (PD) JangSukong (JS) TaeKwangkong (TK) SinPalDalkong 2 (SP2) GeumGangkong (GG) JinPumkong 2 (JP2) PungSanNaMulkong (PS) DaeWon (DW) SoMyeongNaMulkong (SN) SeonHeukkong (SE) Sillogkong (SL) DaePung (DP) AnPyeong (AP) ShinGi (SG) CheongJa 3 (CJ3) DaeMang 2 (DM2) SinHwa (SH)
Landrace Landrace Landrace Landrace Landrace Landrace Landrace 1969 1977 1978 1984 1985 1989 1991 1992 1995 1996 1996 1997 1998 1998 2000 2002 2002 2003 2004 2005 2007
100-seed weight (g) 11.79 8.89 8.40 30.31 19.56 18.44 15.36 16.41 16.80 15.81 15.17 10.19 17.23 19.60 18.61 19.09 17.37 12.01 22.74 8.32 27.60 22.16 18.38 9.96 21.24 33.33 21.45 11.66
Seed coat color Black with Green Black Green Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Black Yellow Yellow Yellow Yellow Black Light-green Yellow
Utilization Cooking with rice Bean sprout Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Bean sprout Soy sauce and tofu Bean sprout Cooking with rice Vegetable Soy sauce and tofu Bean sprout Soy sauce and tofu Cooking with rice Soy sauce and tofu Bean sprout
Crude lipid (%) 17.6de 14.8j 15.0j 17.5def 18.1 cd 17.6de 18.9b 17.6de 14.3j 19.4a 17.8cde 17.2efg 17.5ef 18.5b 18.6bc 16.9fgh 17.4efg 18.9b 18.4bcd 17.0 fg 18.6bc 16.5 h 18.2bcd 17.6cde 15.5i 16.9gh 18.0bc 15.3i
Differences were statistically significant (DMRT, P < 0.05).
wileyonlinelibrary.com/jsfa
© 2014 Society of Chemical Industry
J Sci Food Agric (2014)
Tocopherol content and antioxidant activity in seeds and germinating soybean
www.soci.org
Table 2. Contents of different tocopherol isomers in seeds of soybean cultivars Cultivar SR SM OL HN JD CB GD GK BC JY BU PD JS TK SP2 GG JP2 PS DW SN SE SL DP AP SG CJ3 DM2 SH Cultivar
𝛼-T 86.8 ± 2.5bc 85.8 ± 0.7c 87.8 ± 1.2b 91.2 ± 0.7a 68.8 ± 8.7d 21.3 ± 1.3 ml 33.3 ± 0.7 h 28.9 ± 3.0i 18.2 ± 0.6n 29.4 ± 0.7i 13.7 ± 0.4p 40.7 ± 1.3f 25.3 ± 1.4j 22.9 ± 0.3kjl 22.0 ± 0.7kml 25.2 ± 0.8j 35.8 ± 3.0 g 17.5 ± 1.6o 23.4 ± 0.4kjl 17.0 ± 0.7o 20.5 ± 1.2 m 19.6 ± 0.2 m 21.3 ± 1.5 ml 60.9 ± 4.2e 19.0 ± 2.4 m 21.1 ± 1.0 ml 24.2 ± 1.8kj 35.9 ± 0.4 g *
𝛽-T 18.7 ± 0.6b 18.1 ± 0.2c 18.5 ± 0.3b 19.1 ± 0.1a 15.0 ± 1.4d 3.1 ± 0.3o 5.3 ± 0.1 h 4.2 ± 0.3j 3.7 ± 0.1 m 5.3 ± 0.2 h 2.2 ± 0.1p 4.7 ± 0.2i 3.4 ± 0.2n 3.9 ± 0.8 k 2.4 ± 0.1p 4.2 ± 0.5j 6.6 ± 0.5f 2.1 ± 0.2p 4.4 ± 0.1ij 2.5 ± 0.1p 4.2 ± 0.3j 3.4 ± 0.1mn 3.0 ± 0.2o 7.4 ± 0.6e 1.2 ± 0.1q 3.9 ± 0.2 k 3.8 ± 0.3lk 6.1 ± 0.1 g *
𝛾-T 253.8 ± 0.2bc 255.5 ± 1.4bc 260.8 ± 4.5ab 269.6 ± 0.5a 225.6 ± 16.2def 245.9 ± 13.7c 231.2 ± 3.1de 220.1 ± 8.9ef 227.3 ± 7.0def 230.7 ± 7.3de 150.0 ± 9.8n 217.5 ± 6.8f 225.1 ± 11.7def 235.1 ± 3.5d 257.4 ± 5.8b 209.4 ± 4.9 g 180.1 ± 11.4ij 197.8 ± 15.1 h 163.0 ± 2.5 k 225.4 ± 7.4def 177.7 ± 3.6ij 152.0 ± 1.6 m 193.8 ± 11.9 h 171.2 ± 10.5j 148.4 ± 7.0n 182.6 ± 8.6i 197.5 ± 8.2 h 160.7 ± 1.7 l
𝛿-T
T
116.2 ± 0.5c 117.1 ± 0.8c 119.4 ± 1.5b 123.1 ± 0.6ab 103.8 ± 7.1f 117.1 ± 6.6c 120.2 ± 1.3b 96.3 ± 5.3 g 127.4 ± 3.8a 119.8 ± 4.0b 75.2 ± 2.4 k 69.3 ± 2.0 l 90.8 ± 4.3 h 113.4 ± 1.3d 97.0 ± 1.9 g 106.6 ± 9.0f 82.6 ± 5.1ij 76.6 ± 5.6 k 78.4 ± 1.2 k 110.2 ± 3.5e 104.4 ± 2.2f 76.1 ± 0.9 l 75.8 ± 4.6 k 52.8 ± 3.1n 35.3 ± 1.2o 74.8 ± 3.5 k 85.9 ± 3.6i 63.0 ± 0.7 m
475.5 ± 2.4b 476.5 ± 3.0b 486.6 ± 7.5b 503.1 ± 1.1a 413.3 ± 13.3c 397.7 ± 18.8d 390 ± 3.7de 349.5 ± 9.2f 376.5 ± 11.5e 385.1 ± 12.1de 244.0 ± 7.8n 332.2 ± 10.2 g 344.5 ± 17.5 fg 375.3 ± 4.9e 378.9 ± 8.4e 345.4 ± 13.3 fg 305.1 ± 19.7hi 294.0 ± 29.4i 269.3 ± 4.1 k 355.1 ± 11.7f 306.7 ± 7.0hi 251.2 ± 2.7 m 293.9 ± 18.2i 292.2 ± 18.3i 203.9 ± 10.1o 282.5 ± 13.3j 311.5 ± 13.8 h 265.6 ± 2.7 l
*
*
*
Data represent the means of three replicates ± SD (μg g−1 fresh weight). 𝛼-T, alpha-tocopherol; 𝛽-T, beta-tocopherol; 𝛾-T, gamma-tocopherol; 𝛿-T, delta-tocopherol; T, total tocopherol. * Differences were statistically significant (DMRT, P < 0.05).
the National Institute of Crop Science, Rural Development Administration (RDA) Farm, Suwon, Korea. Soybeans were stored at −70 ∘ C for 1 month until analyzed. Soybean seeds were germinated through the following process. Seeds were soaked in 20% sodium hypochlorite for 12 min at room temperature and thoroughly rinsed three times with distilled water after soaking the seeds in 75% ethanol for 1 min and rinsing twice with distilled water. Seeds were then germinated in plastic dishes containing 15 mL distilled water at 27 ∘ C for 72 h and stored at −70 ∘ C for safe preservation until laboratory extraction and analysis. Extraction method for soybean seeds Soybean seeds and germinating seeds were ground at 260 g for 200 s using an auto-mill disintegrator (Tokken, Japan) in a liquid nitrogen environment. Oil content was analyzed by AOAC method 920.85 (AOAC, 1990)19 using the Soxtherm automatic system (Gerhardt, Germany). Ground soybean (2 g) was packed in a thimble and the oils were extracted with hexane for 3 h. For the analysis of the tocopherol content in soybean seeds, the rapid Soxhlet extraction method was used.20 Finely ground soybeans (2 g) were placed in a Soxtherm automatic extraction unit (Gerhardt, Germany) and extracted with 140 mL n-hexane:EtOAc (85:15, v/v, containing 0.1 g L−1 BHT) at 180 ∘ C for 110 min under J Sci Food Agric (2014)
dim light conditions. Crude lipid extracts were diluted to 50 mL and passed through a 0.2 μm filter unit (Millipore, Billerica, MA, USA) prior to HPLC analysis.20 For seed germination, the direct extraction method was modified.21 Germinating seeds (1 g) were transferred to 5 mL pyrogallol in ethanol (1 g 100 mL−1 ). The extracts were vortexed for 30 s and centrifuged at 6225 × g for 2 min to remove large pieces of debris. The supernatant was collected three times and diluted to a final volume of 10 mL. Aliquots (1 mL) of the supernatant were evaporated with nitrogen gas. Dried samples were resuspended in n-hexane and filtered through a 0.2 μm filter (Millipore). All extracts were performed with three independent samples per cultivars. Lipid peroxidation Soybean seeds and 3-day-old germinating seeds were collected and lipid peroxidation was determined by estimating malondialdehyde content (MDA) using the thiobarbituric acid-reactive substances (TBARS) assay.22 Tissue samples (0.5 g) were macerated in 5 mL of 0.1% trichloroacetic acid and the resulting homogenate was centrifuged (24 903 × g) for 10 min. The aliquots (1 mL) were added with 4 mL of 20% trichloroacetic acid containing 0.5% TBA. The mixture was mixed vigorously, heated at 95 ∘ C for 30 min, cooled rapidly on ice and centrifuged (11 971 × g) for
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/jsfa
www.soci.org 10 min. Absorbance of the supernatant was measured at 532 nm. Measurements were corrected for non-specific turbidity by subtracting the absorbance at 600 nm. The MDA content was calculated using an extinction coefficient of 157 mmol L−1 cm−1 . DPPH and ABTS radical scavenging activity Antioxidant activities of the extracts from seeds and germinating seeds were determined from the scavenging activity of stable DPPH free radicals and the radical cation ABTS+ using a modification of a previously described method.23,24 In a 96-well microtiter plate, soybean extracts (100 μL) were added to 150 μL DPPH in ethanol solution (1.5 × 10−4 mol) and then incubated at 37 ∘ C for 30 min. The absorbance of each solution was measured at 518 nm using a microplate reader (Infinite® 200; Tecan, Madison, WI, USA). Radical scavenging was calculated as (Ac − At /Ac ) × 100, where At and Ac represent absorbance readings with and without sample extracts, respectively. The ABTS assay is based upon the ability of antioxidants to scavenge the radical cation ABTS+ . The radical cation was generated by mixing ABTS stock solution (7.0 × 10−3 mol) with 2.45 × 10−3 mol potassium persulfate. The reaction mixture was diluted with ethanol and the absorbance was determined at 734 nm. About 200 μL diluted ABTS+ solution and 100 μL sample were mixed for 45 s, after incubation at 30 ∘ C for 5 min in a microplate reader. The absorbance was measured immediately after incubation. The ABTS radical cation scavenging activity of each extract was calculated by determining the decrease in absorbance using the following equation: (Ac − At /Ac ) × 100, where At and Ac represent absorbance readings with and without sample extracts, respectively. Preparation of standards For identification and quantification purposes, standard stock solutions of four tocopherol isomers (𝛼-, 𝛽-, 𝛾- and 𝛿-tocopherol)
YY Lee et al.
and four tocotrienol isomers (𝛼-, 𝛽-, 𝛾- and 𝛿-tocotrienol) were prepared in n-hexane (containing 0.1 g L−1 BHT) and stored under nitrogen at −20 ∘ C in the dark. These solutions were diluted in n-hexane:isopropanol (99:1, v/v) prior to analysis. The working solution for each compound was prepared to a concentration ranging from 0.1 to 10 μg mL−1 . Tocopherol and tocotrienol peaks were identified by comparing the sample retention times with the standards. Purity and stability were confirmed by spectrophotometry (Hitachi High-Technologies, Tokyo, Japan) using the known extinction coefficient of each isomer. Concentrations were calculated from the peak areas using linear regression. Statistical analysis The results were statistically evaluated using ANOVA with SAS version 9.2 (SAS Institute, Cary, NC, USA). The means were compared using Duncan tests with a significance level of
Revised: 26 September 2014
Accepted article published: 17 October 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6963
A correlation between tocopherol content and antioxidant activity in seeds and germinating seeds of soybean cultivars Yu Young Lee,a† Hyang Mi Park,a† Tae Young Hwang,a Sun Lim Kim,a Mi Jung Kim,a Seuk Ki Lee,a Min Jung Seo,a Kee Jong Kim,a Young-Up Kwon,a Sang Chul Leeb and Yul Ho Kima* Abstract BACKGROUND: Tocopherols are crucial lipid-soluble antioxidants and essential nutrients. There is increasing interest in the biofortification of crops with vitamin E for reducing micronutrient malnutrition. However, relatively little is known about the development of soybean cultivars with high levels of tocopherol through combined breeding. RESULT: Tocopherol contents of seeds and germinating seeds of 28 Korean soybean cultivars were analyzed and evaluated for health-promoting activities. Total tocopherol concentrations ranged from 203.9 to 503.1 𝛍g g−1 in seeds and from 20.1 to 230.1 𝛍g g−1 in germinating seeds. The traditional landraces of HaNagari (HN, 503.1 𝛍g g−1 ), Orialtae (OL, 486.6 𝛍g g−1 ), SuMoktae (SM, 476.5 𝛍g g−1 ) and SoRitae (SR, 475.5 𝛍g g−1 ) showed high levels of tocopherol content. The contents of the four isomers of tocopherol in seeds and germinating seeds were correlated with lipid peroxidation. The 𝜸- and 𝜹-tocopherol contents in seeds were related to 1,1-diphenyl-2-picrylhydrazyl free radical scavenging activity (0.434; P < 0.01 and 0.373; P < 0.05). CONCLUSION: Total tocopherol content was higher in soybean landraces as compared with modern cultivars developed by cross-breeding. These results suggest that soybean breeding is necessary to increase tocopherol levels. © 2014 Society of Chemical Industry Keywords: antioxidant activity; lipid peroxidation; seed; germination; soybean; tocopherols
INTRODUCTION Tocopherols, generally known as vitamin E, are crucial lipid-soluble antioxidants. Vitamin E consists of a polar chromanol ring linked to a 15-carbon tail and comprises eight chemically distinct compounds that are separated into tocopherols and tocotrienols according to hydrophobic tail saturation. Tocopherols contain a fully saturated tail, whereas tocotrienols have three unsaturated double bonds. The different tocopherol and tocotrienol isomers, i.e. 𝛼, 𝛽, 𝛾 and 𝛿, are distinguished by the locations of methyl groups on the chromanol ring.1,2 Tocopherols play a significant role in food preservation and disease prevention.3 It is commonly believed that the beneficial effects of vitamin E are derived from preventing the breakdown of polyunsaturated fatty acids in membrane lipids and influencing cellular responses to oxidative stress in signal transduction pathways.4 Soybean (Glycine max L.) is an important highland crop consumed in various forms such as soybean oil, soybean sprout, soy paste, soymilk and tofu.5,6 Soybean oil corresponds to 20–30% of the vegetable oils world market and is an important source of natural tocopherols.7,8 Soybean sprouts, in particular, have been consumed as a year-round vegetable for thousands of years in Korea. There are excellent sources of nutrients and vitamins.9 Annually, more than 500 000 tons of soybean sprouts are consumed as vegetable in soups, salads and side dishes in Korea.10 At present, the J Sci Food Agric (2014)
market value for soybean sprouts is around $700 million and the consumption levels have been greatly increased by the diversification of cooking methods and the creation of new demand.11,12 However, relatively little is known about the impact of soybean germination on tocopherols. Many published papers have investigated tocopherol concentrations in soybean. The range and mean contents of four tocopherol isomers in seeds of five cultivars (DaewonKong, Taekwangkong, Jinpumkong, Hwaseongputkong and Ilpumgumjeongkong) was reported to be 305–389 μg g−1 dry weight.13 Five cultivars of black soybeans contained 249–405 μg g−1 of 𝛾-tocopherol that underwent significant degradation within a month of storage at room temperature (P < 0.01) but remained stable for 6 months at 4 ∘ C
∗
Correspondence to: Yul Ho Kim, National Institute of Crop Science, Rural Development Administration, Suwon 441-857, Republic of Korea. E-mail: [email protected]
† These authors contributed equally to this work. a National Institute of Crop Science, Rural Development Administration, Suwon, 441-857, Republic of Korea b School of Applied Bioscience, College of Agriculture and Life Sciences, Kyungpook National University, Daegu, 702-701, Republic of Korea
www.soci.org
© 2014 Society of Chemical Industry
www.soci.org and 14 months at −80 ∘ C.14 The tocopherol contents of soybean germplasms originating from different countries (USA, China and Korea) were also investigated in addition to seed size on the basis of 100-seed weight. Among Chinese soybeans, 13–24g of 100-seed weight group had the highest (256.1 μg g−1 ) average total tocopherol content.15 In the last decade, a complementary approach for development of micronutrient-dense crops. termed biofortification, has been undertaken with the aim of reducing micronutrient malnutrition and preventing diseases using genetic engineering approaches. Significant progress has been made in the development of crops biofortified with vitamin E and altered tocopherol or tocotrienol compositions.16 – 18 To date, no attempts have been made to develop a soybean cultivar with a high level of vitamin E using combined breeding in Korea. In this study, the concentrations of tocopherols in seeds and germinating seeds of 28 soybean cultivars were determined to find a tocopherol-rich soybean for biofortification. In addition, the relationship between the concentration of tocopherol isomers and antioxidant activities using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′ -azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) assay and lipid peroxidation inhibition test was investigated. The association between vitamin E isomer content and antioxidant activity was investigated in soybean seeds and germinating seeds.
YY Lee et al.
MATERIALS AND METHODS Chemicals and Instruments Ethanol, butylated hydroxytoluene (BHT), DPPH, ABTS, potassium persulfate, trichloroacetic acid and tocopherols standards were obtained from Sigma Chemical Co. (St Louis, MO, USA). Ethyl acetate (EtOAc), n-hexane, isopropanol and water were purchased from JT Baker (Phillipsburg, NJ, USA). All solvents and reagents used were analytical grade. Thiobarbituric acid (TBA) was supplied by MP Biomedicals, while tocotrienols were purchased from Davos Life Science (Tuas, Singapore). The tocopherol and tocotrienol contents of soybeans were determined by normal-phase high-performance liquid chromatography (HPLC). The HPLC system consisted of a solvent delivery pump (model 515; Waters, Milford, MA, USA) equipped with a spectrofluorometric detector (model 2475, Waters) and a Lichrosorb Si60 column (4.6 × 250 mm, 5 μm; Hibar, Darmstadt, Germany). The isocratic mobile phase was n-hexane:isopropanol (99:1, v/v) at a flow rate of 1.5 mL min−1 . Excitation and emission spectra were 290 and 330 nm, respectively. The injection volume was 10 μL and the column temperature was regulated at 30 ∘ C. Samples Twenty-eight cultivars of soybean were used in the experiment (Table 1). All soybeans were harvested on 24 October 2011 at
Table 1. Characteristics of 28 soybean cultivars Cultivar
Registration year
SoRitae (SR) SuMoktae (SM) Orialtae (OL) HaNagari (HN) JangDanBaemok (JD) ChungBukbaek (CB) GwangDu (GD) GwangKyo (GK) BaegCheon (BC) JangYeopkong (JY) BaekUnkong (BU) PalDalkong (PD) JangSukong (JS) TaeKwangkong (TK) SinPalDalkong 2 (SP2) GeumGangkong (GG) JinPumkong 2 (JP2) PungSanNaMulkong (PS) DaeWon (DW) SoMyeongNaMulkong (SN) SeonHeukkong (SE) Sillogkong (SL) DaePung (DP) AnPyeong (AP) ShinGi (SG) CheongJa 3 (CJ3) DaeMang 2 (DM2) SinHwa (SH)
Landrace Landrace Landrace Landrace Landrace Landrace Landrace 1969 1977 1978 1984 1985 1989 1991 1992 1995 1996 1996 1997 1998 1998 2000 2002 2002 2003 2004 2005 2007
100-seed weight (g) 11.79 8.89 8.40 30.31 19.56 18.44 15.36 16.41 16.80 15.81 15.17 10.19 17.23 19.60 18.61 19.09 17.37 12.01 22.74 8.32 27.60 22.16 18.38 9.96 21.24 33.33 21.45 11.66
Seed coat color Black with Green Black Green Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Yellow Black Yellow Yellow Yellow Yellow Black Light-green Yellow
Utilization Cooking with rice Bean sprout Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Soy sauce and tofu Bean sprout Soy sauce and tofu Bean sprout Cooking with rice Vegetable Soy sauce and tofu Bean sprout Soy sauce and tofu Cooking with rice Soy sauce and tofu Bean sprout
Crude lipid (%) 17.6de 14.8j 15.0j 17.5def 18.1 cd 17.6de 18.9b 17.6de 14.3j 19.4a 17.8cde 17.2efg 17.5ef 18.5b 18.6bc 16.9fgh 17.4efg 18.9b 18.4bcd 17.0 fg 18.6bc 16.5 h 18.2bcd 17.6cde 15.5i 16.9gh 18.0bc 15.3i
Differences were statistically significant (DMRT, P < 0.05).
wileyonlinelibrary.com/jsfa
© 2014 Society of Chemical Industry
J Sci Food Agric (2014)
Tocopherol content and antioxidant activity in seeds and germinating soybean
www.soci.org
Table 2. Contents of different tocopherol isomers in seeds of soybean cultivars Cultivar SR SM OL HN JD CB GD GK BC JY BU PD JS TK SP2 GG JP2 PS DW SN SE SL DP AP SG CJ3 DM2 SH Cultivar
𝛼-T 86.8 ± 2.5bc 85.8 ± 0.7c 87.8 ± 1.2b 91.2 ± 0.7a 68.8 ± 8.7d 21.3 ± 1.3 ml 33.3 ± 0.7 h 28.9 ± 3.0i 18.2 ± 0.6n 29.4 ± 0.7i 13.7 ± 0.4p 40.7 ± 1.3f 25.3 ± 1.4j 22.9 ± 0.3kjl 22.0 ± 0.7kml 25.2 ± 0.8j 35.8 ± 3.0 g 17.5 ± 1.6o 23.4 ± 0.4kjl 17.0 ± 0.7o 20.5 ± 1.2 m 19.6 ± 0.2 m 21.3 ± 1.5 ml 60.9 ± 4.2e 19.0 ± 2.4 m 21.1 ± 1.0 ml 24.2 ± 1.8kj 35.9 ± 0.4 g *
𝛽-T 18.7 ± 0.6b 18.1 ± 0.2c 18.5 ± 0.3b 19.1 ± 0.1a 15.0 ± 1.4d 3.1 ± 0.3o 5.3 ± 0.1 h 4.2 ± 0.3j 3.7 ± 0.1 m 5.3 ± 0.2 h 2.2 ± 0.1p 4.7 ± 0.2i 3.4 ± 0.2n 3.9 ± 0.8 k 2.4 ± 0.1p 4.2 ± 0.5j 6.6 ± 0.5f 2.1 ± 0.2p 4.4 ± 0.1ij 2.5 ± 0.1p 4.2 ± 0.3j 3.4 ± 0.1mn 3.0 ± 0.2o 7.4 ± 0.6e 1.2 ± 0.1q 3.9 ± 0.2 k 3.8 ± 0.3lk 6.1 ± 0.1 g *
𝛾-T 253.8 ± 0.2bc 255.5 ± 1.4bc 260.8 ± 4.5ab 269.6 ± 0.5a 225.6 ± 16.2def 245.9 ± 13.7c 231.2 ± 3.1de 220.1 ± 8.9ef 227.3 ± 7.0def 230.7 ± 7.3de 150.0 ± 9.8n 217.5 ± 6.8f 225.1 ± 11.7def 235.1 ± 3.5d 257.4 ± 5.8b 209.4 ± 4.9 g 180.1 ± 11.4ij 197.8 ± 15.1 h 163.0 ± 2.5 k 225.4 ± 7.4def 177.7 ± 3.6ij 152.0 ± 1.6 m 193.8 ± 11.9 h 171.2 ± 10.5j 148.4 ± 7.0n 182.6 ± 8.6i 197.5 ± 8.2 h 160.7 ± 1.7 l
𝛿-T
T
116.2 ± 0.5c 117.1 ± 0.8c 119.4 ± 1.5b 123.1 ± 0.6ab 103.8 ± 7.1f 117.1 ± 6.6c 120.2 ± 1.3b 96.3 ± 5.3 g 127.4 ± 3.8a 119.8 ± 4.0b 75.2 ± 2.4 k 69.3 ± 2.0 l 90.8 ± 4.3 h 113.4 ± 1.3d 97.0 ± 1.9 g 106.6 ± 9.0f 82.6 ± 5.1ij 76.6 ± 5.6 k 78.4 ± 1.2 k 110.2 ± 3.5e 104.4 ± 2.2f 76.1 ± 0.9 l 75.8 ± 4.6 k 52.8 ± 3.1n 35.3 ± 1.2o 74.8 ± 3.5 k 85.9 ± 3.6i 63.0 ± 0.7 m
475.5 ± 2.4b 476.5 ± 3.0b 486.6 ± 7.5b 503.1 ± 1.1a 413.3 ± 13.3c 397.7 ± 18.8d 390 ± 3.7de 349.5 ± 9.2f 376.5 ± 11.5e 385.1 ± 12.1de 244.0 ± 7.8n 332.2 ± 10.2 g 344.5 ± 17.5 fg 375.3 ± 4.9e 378.9 ± 8.4e 345.4 ± 13.3 fg 305.1 ± 19.7hi 294.0 ± 29.4i 269.3 ± 4.1 k 355.1 ± 11.7f 306.7 ± 7.0hi 251.2 ± 2.7 m 293.9 ± 18.2i 292.2 ± 18.3i 203.9 ± 10.1o 282.5 ± 13.3j 311.5 ± 13.8 h 265.6 ± 2.7 l
*
*
*
Data represent the means of three replicates ± SD (μg g−1 fresh weight). 𝛼-T, alpha-tocopherol; 𝛽-T, beta-tocopherol; 𝛾-T, gamma-tocopherol; 𝛿-T, delta-tocopherol; T, total tocopherol. * Differences were statistically significant (DMRT, P < 0.05).
the National Institute of Crop Science, Rural Development Administration (RDA) Farm, Suwon, Korea. Soybeans were stored at −70 ∘ C for 1 month until analyzed. Soybean seeds were germinated through the following process. Seeds were soaked in 20% sodium hypochlorite for 12 min at room temperature and thoroughly rinsed three times with distilled water after soaking the seeds in 75% ethanol for 1 min and rinsing twice with distilled water. Seeds were then germinated in plastic dishes containing 15 mL distilled water at 27 ∘ C for 72 h and stored at −70 ∘ C for safe preservation until laboratory extraction and analysis. Extraction method for soybean seeds Soybean seeds and germinating seeds were ground at 260 g for 200 s using an auto-mill disintegrator (Tokken, Japan) in a liquid nitrogen environment. Oil content was analyzed by AOAC method 920.85 (AOAC, 1990)19 using the Soxtherm automatic system (Gerhardt, Germany). Ground soybean (2 g) was packed in a thimble and the oils were extracted with hexane for 3 h. For the analysis of the tocopherol content in soybean seeds, the rapid Soxhlet extraction method was used.20 Finely ground soybeans (2 g) were placed in a Soxtherm automatic extraction unit (Gerhardt, Germany) and extracted with 140 mL n-hexane:EtOAc (85:15, v/v, containing 0.1 g L−1 BHT) at 180 ∘ C for 110 min under J Sci Food Agric (2014)
dim light conditions. Crude lipid extracts were diluted to 50 mL and passed through a 0.2 μm filter unit (Millipore, Billerica, MA, USA) prior to HPLC analysis.20 For seed germination, the direct extraction method was modified.21 Germinating seeds (1 g) were transferred to 5 mL pyrogallol in ethanol (1 g 100 mL−1 ). The extracts were vortexed for 30 s and centrifuged at 6225 × g for 2 min to remove large pieces of debris. The supernatant was collected three times and diluted to a final volume of 10 mL. Aliquots (1 mL) of the supernatant were evaporated with nitrogen gas. Dried samples were resuspended in n-hexane and filtered through a 0.2 μm filter (Millipore). All extracts were performed with three independent samples per cultivars. Lipid peroxidation Soybean seeds and 3-day-old germinating seeds were collected and lipid peroxidation was determined by estimating malondialdehyde content (MDA) using the thiobarbituric acid-reactive substances (TBARS) assay.22 Tissue samples (0.5 g) were macerated in 5 mL of 0.1% trichloroacetic acid and the resulting homogenate was centrifuged (24 903 × g) for 10 min. The aliquots (1 mL) were added with 4 mL of 20% trichloroacetic acid containing 0.5% TBA. The mixture was mixed vigorously, heated at 95 ∘ C for 30 min, cooled rapidly on ice and centrifuged (11 971 × g) for
© 2014 Society of Chemical Industry
wileyonlinelibrary.com/jsfa
www.soci.org 10 min. Absorbance of the supernatant was measured at 532 nm. Measurements were corrected for non-specific turbidity by subtracting the absorbance at 600 nm. The MDA content was calculated using an extinction coefficient of 157 mmol L−1 cm−1 . DPPH and ABTS radical scavenging activity Antioxidant activities of the extracts from seeds and germinating seeds were determined from the scavenging activity of stable DPPH free radicals and the radical cation ABTS+ using a modification of a previously described method.23,24 In a 96-well microtiter plate, soybean extracts (100 μL) were added to 150 μL DPPH in ethanol solution (1.5 × 10−4 mol) and then incubated at 37 ∘ C for 30 min. The absorbance of each solution was measured at 518 nm using a microplate reader (Infinite® 200; Tecan, Madison, WI, USA). Radical scavenging was calculated as (Ac − At /Ac ) × 100, where At and Ac represent absorbance readings with and without sample extracts, respectively. The ABTS assay is based upon the ability of antioxidants to scavenge the radical cation ABTS+ . The radical cation was generated by mixing ABTS stock solution (7.0 × 10−3 mol) with 2.45 × 10−3 mol potassium persulfate. The reaction mixture was diluted with ethanol and the absorbance was determined at 734 nm. About 200 μL diluted ABTS+ solution and 100 μL sample were mixed for 45 s, after incubation at 30 ∘ C for 5 min in a microplate reader. The absorbance was measured immediately after incubation. The ABTS radical cation scavenging activity of each extract was calculated by determining the decrease in absorbance using the following equation: (Ac − At /Ac ) × 100, where At and Ac represent absorbance readings with and without sample extracts, respectively. Preparation of standards For identification and quantification purposes, standard stock solutions of four tocopherol isomers (𝛼-, 𝛽-, 𝛾- and 𝛿-tocopherol)
YY Lee et al.
and four tocotrienol isomers (𝛼-, 𝛽-, 𝛾- and 𝛿-tocotrienol) were prepared in n-hexane (containing 0.1 g L−1 BHT) and stored under nitrogen at −20 ∘ C in the dark. These solutions were diluted in n-hexane:isopropanol (99:1, v/v) prior to analysis. The working solution for each compound was prepared to a concentration ranging from 0.1 to 10 μg mL−1 . Tocopherol and tocotrienol peaks were identified by comparing the sample retention times with the standards. Purity and stability were confirmed by spectrophotometry (Hitachi High-Technologies, Tokyo, Japan) using the known extinction coefficient of each isomer. Concentrations were calculated from the peak areas using linear regression. Statistical analysis The results were statistically evaluated using ANOVA with SAS version 9.2 (SAS Institute, Cary, NC, USA). The means were compared using Duncan tests with a significance level of
