
Soybeans (
Soybean isoflavones are structurally diverse and found in aglycone (daidzein, genistein, glycitein),
It is widely documented that the contents of antho-cyanins and isoflavones in soybeans are affected by seed-related agronomical characters and environmental factors (Hoeck
In Korea, black soybeans are largely consumed and many researchers determined the levels of dietary isoflavones and anthocyanins in their seeds. Moreover, the influences of environmental factors such as place of cultivation, planting dates and growing season, and seed-related characters such as seed coat color, maturity and seed weight have been investigated (Lee
Anthocyanin standards including D-3-
The 323 black soybean landraces of Korean origin were obtained from the Gene bank of the National Agrobiodiversity Center, Rural Development Administration (RDA, Jeonju, Korea). The soybeans were directly sown at a spacing of 90 cm in an experimental field located at the center on June 5, 2018. All landraces were cultivated under similar conditions and the average temperature and precipitation during the cropping period are shown in Table 1. The change in pod color was used as an index of maturity, and seeds were harvested when 95% of their pods attained matured color (Zhou
Table 1 . Average temperature and precipitation of the cultivation area during the cropping period (2018).
Parameter | Cultivation period | Harvest period | ||||||
---|---|---|---|---|---|---|---|---|
June | July | August | September | Early-October (1-10) | Mid-October (11-20) | Late-October (21-31) | ||
Average temperature (℃) | 23.1 | 27.8 | 28.6 | 21.7 | 16.1 | 13 | 12.1 | |
Average precipitation (mm) | 137.2 | 169.1 | 368.9 | 101.8 | 98.7 | 0.9 | 23.4 |
Isoflavones were extracted using the method developed by Nawaz
Identification and quantification of the target isoflavones and anthocyanins were achieved using the corresponding external standards. Analysis was conducted on a Nanospace SI-2 Semi-microcolumn HPLC system (Shiseido, Tokyo, Japan) coupled to a UV-Vis detector. Isoflavones were eluted on a Shiseido UG-120 column (4.6 × 250 mm; 5 µm) which was maintained at 30℃ while anthocyanins were separated on a Shiseido C18 column type MGII (4.6 × 250 mm; 5 µm) which was maintained at 40℃. During isoflavone analysis, a binary solvent system composed of water (A) and acetonitrile (B) was used as a mobile phase. The gradient elution was started with 15%B for the first 10 minutes followed by an increase to 20%B for another 10 minutes, and to 50% for 20 minutes. Then, the mobile phase was equilibrated to 15%B for the last 10 minutes. The solvent flow rate was 0.8 mL/minute throughout the analysis and the sample injection volume was 10 µL. The isoflavones were detected at 260 nm wavelength in the acquired chromatograms. During anthocyanin analysis, the mobile phase was composed of water (A) and acetonitrile (B) each containing 5% formic acid. The elusion was started with 10%B followed by an increase to 15%B at 0.8 mL/minute flow rate until 10 minutes, then to 46%B at 0.8 mL/minute until 15.3 minutes, and to 90%B at 1 mL/minute until 17 minutes. Finally, the solvent was equilibrated to 10%B at 0.8 mL/minute until 24 minutes. The sample injection volume was 20 µL and the anthocyanins were read at 520 nm wavelength.
All measurements in the present study were carried out in duplicates, and results were expressed as mean ± standard deviation (SD) on a dry weight basis. One-way analysis of variance was computed using xlstat-software (Addinsoft, NY, USA) and applied to statistically determine the difference between treatments. Box plots, principal component and Pearson correlation analysis were performed on R-software version 4.0.2 (http://www.r-project.org/).
The individual isoflavones and anthocyanins were identified and quantified using the corresponding external standards as described before. All five isoflavones (daidzin, glycitin, genistin, malonyldaidzin, and malonylgensitin) were detected in every landrace. With respect to individual anthocyanins, however, variations were observed for few landraces. C-3-
The soybean landraces showed variations in both isoflavone and anthocyanin contents (Supplementary Table S1). The total isoflavone (TIC) and total anthocyanin (TAC) contents were in the ranges of 491.29-1998.39 µg/g and 452.60-2789.37 mg/100 g with means of 1447.87 µg/g and 1547.64 mg/100 g, respectively. Compared to the TIC, the TAC had a higher coefficient of variation (%CV) (Supplementary Table S2). The highest TIC was found in landrace S25 (IT: 121504) and was approximately 4-times higher than the lowest TIC found in landrace S84 (IT: 177871) (
The box plots in Fig. 2 show the variations in isoflavone and anthocyanin contents among the green and yellow cotyledon black soybeans. The corresponding numerical values can be viewed in Supplementary Table S2. The average TIC was higher in green cotyledon soybeans (1499.93 µg/g) than in yellow cotyledon soybeans (1423.09 µg/g). Besides, all individual isoflavones, except glycitin, followed a similar pattern of variation as the TIC each being higher in green cotyledon soybeans than in yellow cotyledon soybeans. Among the five isoflavones, the level of malonylgenistin was the highest in each class. The average TAC followed a comparable pattern as the TIC and was higher in green cotyledon soybeans (1656.05 mg/100 g) than in yellow cotyledon soybeans (1489.30 mg/100 g). Among individual anthocyanins, C-3-
The relationship between harvest periods (Early-October, Mid-October and Late-October) and isoflavone and anthocyanin contents are displayed in Fig. 3. The corresponding numerical values can be viewed in Supplementary Table S2. The average TIC was the highest in Late-October (1517.31 µg/g) followed by Mid-October (1454.54 µg/g) and Early-October (1340.95 mg/g). Among individual isoflavones, the average genistin and malonylgenistin contents were the highest in Late-October (166.84 and 828.72 mg/g, respectively) followed by Mid-October (147.31 and 764.92 mg/g, respectively) and Early-October (129.66 and 741.95 mg/g, respectively). Besides, the average daidzin and malonyldaidzin contents were in the order of Mid-October (98.26 and 412.02 mg/g, respectively) > Late-October (92.81 and 400.29 mg/g, respectively) > Early-October (82.26 and 356.01 mg/g, respectively) while the average glycitin content was in the order of Mid-October (32.03 mg/g) > Early-October (31.08 mg/g) > Late-October (28.63 mg/g). With respect to anthocyanins, the average TAC followed a similar pattern of variation as the TIC and was the highest in Late-October (1765.76 mg/100 g) than in Mid-October (1503.93 mg/100 g) and Early-October (1350.91 mg/100 g). Moreover, the average content of the principal anthocyanin, C-3-
The pairwise association between isoflavones and anthocyanins in the entire soybean landraces was computed using Pearson correlation analysis (Table 2). The TIC was positively correlated with all the individual isoflavones, and all associations were significant. The TIC showed a strong association with malonylglycitin (
Table 2 . Pearson correlation coefficient (
Variables | Daidzin | Glycitin | Genistin | Malonyl-daidzin | Malonyl-genistin | TIC | D-3- | C-3- | Pt-3- |
---|---|---|---|---|---|---|---|---|---|
Glycitin | 0.27**** | ||||||||
Genistin | 0.69**** | 0.22**** | |||||||
Malonyldaidzin | 0.61**** | 0.08ns | 0.25**** | ||||||
Malonylgenistin | 0.22**** | 0.01ns | 0.43**** | 0.57**** | |||||
TICz) | 0.58**** | 0.14* | 0.59**** | 0.81**** | 0.90**** | ||||
D-3- | 0.04ns | 0.13* | 0.02ns | 0.02ns | ‒0.04ns | ‒0.01ns | |||
C-3- | ‒0.02ns | ‒0.16** | 0.12* | 0.01ns | 0.20*** | 0.17** | ‒0.05ns | ||
Pt-3- | 0.03ns | 0.11* | 0.19** | 0.01ns | 0.14* | 0.13* | 0.40**** | ‒0.14* | |
TACv) | ‒0.01ns | ‒0.12* | 0.14* | 0.11ns | 0.20** | 0.18** | 0.24**** | 0.95**** | 0.05ns |
z)Total isoflavone content; y)Delphinidin-3-
nsNot significant; *
Table 3 . Contributions and eigenvalues of variables in the first four principal components.
Variables | Principal components | |||
---|---|---|---|---|
PC1 | PC2 | PC3 | PC4 | |
Daidzin | 14.88 | 5.61 | 0.06 | 16.36 |
Glycitin | 1.17 | 8.23 | 6.29 | 26.27 |
Genistin | 14.99 | 1.17 | 0.77 | 11.48 |
Malonyldaidzin | 17.93 | 0.56 | 2.64 | 3.31 |
Malonylgenistin | 17.89 | 0.21 | 1.13 | 18.82 |
Total isoflavone content | 26.65 | 0.27 | 1.03 | 4.45 |
Delphinidin-3- | 0.15 | 0.08 | 45.98 | 0.93 |
Cyanidin-3- | 2.49 | 42.39 | 0.08 | 4.73 |
Petunidin-3- | 0.87 | 1.17 | 37.95 | 11.17 |
Total anthocyanin content | 2.99 | 40.30 | 4.08 | 2.48 |
Eigenvalue | 3.44 | 2.00 | 1.49 | 1.06 |
Variability (%) | 34.31 | 20.10 | 14.90 | 10.61 |
Cumulative variance (%) | 34.31 | 54.41 | 69.31 | 79.92 |
In this study, 323 black soybean landraces were grown in Korea, the contents of three common anthocyanins (D-3-
The TIC and TAC were in the ranges of 491.29-1998.39 mg/g and 452.60-2789.37 mg/100 g, respectively. Of the entire soybeans analyzed in the present study, 17.03% of the landraces (i.e. 55 landraces) had a TIC of > 1800 mg/g, and out of these 36.36% (i.e. 20 landraces) had a TAC of > 2000 mg/100 g (Supplementary Table S1). Hence, these landraces could be important sources of high isoflavone and anthocyanin concentrations. Previously, many studies determined the TIC and TAC in various Korean black soybeans. Nevertheless, the reported total contents are inconsistent and wide-ranging due to the disparity in the number and identity of target components (Kim
In their early growth seedling, black soybeans develop either green or yellow cotyledon which could later affect the metabolite contents in matured seeds (Eum
In Korea, the fall season (September to November) is the harvesting time for most summer cropping cereals and legumes (Wang and Mauzerall 2004). In this study, the harvest period ranged from the beginning to the end of October. Of the entire 323 soybeans, 72 landraces were harvested in Early-October, 155 landraces in Mid-October, and 96 landraces in Late-October (Fig. 1). The days to maturity was in the ranges of 116-146 days with an average of 122, 130, and 141 days for landraces harvested in Early-October, Mid-October, and Late-October, respectively. Harvest period appeared to cause significant variation in the contents of isoflavones and anthocyanins. Both the average TIC and TAC were the highest in Late-October followed by Mid-October and Early-October. The average TIC in Late-October and Mid-October was significantly different from the TIC in Early-October (
The pair-wise correlation between the different components and the distributions of the soybean landraces were viewed by Pearson correlation and principal component analysis. The TAC and TIC showed strong associations with their respective major components, and the observations were comparable with many previous studies (Bursac
In this study, 323 black soybean landraces of Korean origin were cultivated, the contents of five major isoflavones and three major anthocyanins were determined, and the influences of cotyledon color and harvest period on each was assessed. Green cotyledon soybeans had a higher average TIC and TAC than yellow cotyledon soybeans. Besides, landraces harvested in Late-October displayed significantly high average TIC and TAC than landraces harvested in Mid-October and Early-October. In general, cotyledon color appeared to have a significant effect on the TAC, whereas the harvest period appeared to have a significant effect on both the TAC and TIC. Of all the studied landraces, 20 soybeans including S4 (IT:104314), S56 (IT:177573), S70 (IT:177720), S100 (IT:186183), S103 (IT:201834), S113 (IT:201852), S146 (IT:215908), S148 (IT: 216312), S175 (IT:228789), S191 (IT:242611), S192 (IT:252252), S206 (IT: 263106), S218 (IT:263576), S235 (IT:269626), S245 (IT:269847), S256 (IT:274472), S259 (IT:274511), S297 (IT:278716), S309 (IT:283894), and S315 (IT: 285974) had a TAC of > 2000 mg/100 g and a TIC of > 1800 mg/g. Our findings suggest that these landraces could be important sources of high dietary isoflavone and anthocyanin concentrations. Besides, those landraces harvested in Late-October could be targeted to advance their cultivation in Korea.
This work was supported by the Research Program for Agricultural Science & Technology Development (Project No. PJ 014172032021) of the National Institute of Agricultural Sciences, Rural Development Administration (Jeonju, Republic of Korea).
The authors declare no conflict of interest.
![]() |
![]() |