Isoflavones and soyasaponins in the germ of Korean soybean [Glycine max (L.) Merr.] cultivars and their compound-enhanced BMP-2-induced bone formation

Soybeans are used worldwide as food and as a healthy ingredient. Specifically, soy germ (SG) has received considerable attention owing to its abundant nutritional and biological components. This study aimed to elucidate the contents of isoflavone and soyasaponin of SG in 24 Korean soybean cultivars and the osteogenic activity of individual compounds. The isoflavone content in the SG ranged from 1110.9 to 3131.1 mg/100 g, and the soyasaponin content in SG ranged from 1173.5 to 3582.3 mg/100 g. The isoflavone and soyasaponin content depended on soybean cultivars. All isoflavone and soyasaponin compounds enhanced bone morphogenetic protein-2-mediated osteoblast differentiation in a dose-dependent manner, especially soyasaponin Ab. In conclusion, our results suggest that Seonpung cultivar with high soyasaponin Ab is beneficial for developing functional materials.


Introduction
Soybeans [Glycine max (L.) Merr.] are cultivated worldwide because they are rich in primary metabolites such as proteins and oils. In addition, soybeans contain many secondary metabolites such as isoflavones, soyasaponins and tocopherols [6]. Soybean seeds structurally consist of the seed coat, cotyledon, and germ [14]. The isoflavone and soyasaponin content of the germ is higher than that of the seed coat and cotyledon [1,5,21].
Soyasaponins are oleanane-type triterpenoid saponins. Soyasaponins are divided into soyasaponin A group, B group, E group and DDMP group [10,17,18]. The compounds of the soyasaponin A group are known to exhibit various biological activities such as bone health, antiobesity, and anti-oxidant activities [3,7,15]; the compounds of the soyasaponin B group are known to exhibit various biological activities such as bone health, antiinflammatory, anti-cancer, hepatoprotective and renin inhibitory activities [8,11,13,19,23].
However, until now, the effect of individual isoflavone and soyasaponin compounds on osteoblast differentiation has not been simultaneously studied. Therefore, we determined isoflavone and soyasaponin contents in soy germ (SG) and investigated the effect of isoflavones

Preparation of soybean cultivars, SG and SG extract
Twenty-four soybean [Glycine max (L.) Merr.] cultivars were grown on the experimental field at the National Institute of Crop Science, Jeonbuk, Korea, and harvested in 2018. The separation of SG was conducted using the previously published method [9] with some modifications. Soybean seeds were crushed using a grinder and cotyledon, and the seed coat was removed using a sieve to separate the SG. To make the SG extract, each SG was dried in a freeze-dryer and then ground. Ground SG was defatted using hexane, and the defatted sample (1 g) was extracted using MeOH (40 mL) for 24 h at room temperature. The extract was centrifuged at 5000 rpm for 10 min at 4 °C, and the supernatant was filtered through a regenerated cellulose syringe filter (0.2 μm). The filtered solution was transferred into a 2 mL vial for the analysis of isoflavones and soyasaponins.

Isoflavone analysis
Isoflavone analysis was conducted using an ultra-high performance liquid chromatography (UHPLC, Dionex Ultimate 3000, Thermo Scientific) instrument equipped with a HALO C18 (2.7 μm, 2.1 mm × 100 mm) column. The mobile phases A and B were 0.1% acetic acid in water and 0.1% acetic acid in acetonitrile, respectively. The solvent flow rate was 0.3 mL/min, and the column temperature was set to 35 °C. The gradient was programmed as 0-2 min, 10% B; 35 min, 30% B; 36 min, 90% B; 36-39 min, 90% B; and 40 min, 10% B, held for 5 min before returning to the initial condition. After the injection of 1.3 μL of the sample, eluted isoflavones were detected at 254 nm using a diode array detector (DAD, Thermo Scientific). The calibration curve was plotted by peak area versus the concentration of isoflavones. To prepare the standard stock solution, 12 isoflavones were dissolved in DMSO at the concentration of 1 mg/mL. The stock solution was serially diluted to make the standard solution (3.125, 6.25, 12.5, 25, and 50 μg/mL).

Soyasaponin analysis
Soyasaponins analysis was conducted using a UHPLC (Dionex Ultimate 3000, Thermo Scientific) instrument equipped with an Acclaim TM RSLC Polar Advantage II (2.2 μm, 2.1 mm × 150 mm) column. The mobile phases A and B were 0.1% acetic acid in water and 0.1% acetic acid in acetonitrile, respectively. The solvent flow rate was 0.5 mL/min, and the column temperature was set to 40 °C. The gradient was programmed as 0-1 min, 20% B; 5 min, 30% B; 35 min, 45% B; 40-42 min, 90% B; and 43 min, 20% B, held for 7 min before returning to the initial conditions. After the injection of 1.3 μL of the sample, eluted soyasaponins were detected using a charged aerosol detector (CAD, Corona Veo, Thermo Scientific). The setting for CAD were as follows: gas, nitrogen; power function, 1.3; pressure, 61 psi; filter, 10 s; gain, 100 pA; evaporation temperature, 50 °C; and data collection rate, 10 Hz. The calibration curve was plotted as the peak area versus the concentration of soyasaponins. To prepare the standard stock solution, 10 soyasaponins were dissolved in DMSO at the concentration of 1 mg/mL. The stock solution was serially diluted to make the standard solution (6.25, 12.5, 25, 50, and 100 μg/mL).

Osteoblast cell Culture and differentiation
All cell experiments were performed as previously described [3] with some modifications. Mouse mesenchymal precursor C2C12 cells were purchased from the American Type Collection (Manassas, VA, USA). C2C12 cells were maintained in an alpha minimum essential medium (α-MEM) containing 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% FBS. To differentiate C2C12 into osteoblasts, the cells were seeded and allowed to attach and grow for 1 d; then which the medium was replaced with a differentiation medium (α-MEM containing 5% FBS and 100 ng/mL rhBMP-2). The medium was changed every 3 d.

Table 1 Isoflavone content of soy germ in 24 soybean cultivars
The mean values in the same column indicated by the same letter are not significantly different at the level of 0

Alkaline phosphatases (ALP) staining and activity assay
The ALP activity of C2C12 cells was assessed using ALP staining and an ALP activity detection kit (Sigma-Aldrich, St. Louis, MO, USA). Briefly, C2C12 cells were cultured under osteogenic differentiation conditions in the presence of the vehicle, isoflavones, or soyasaponins. After differentiation for 3 d, the cells were washed twice with PBS, fixed with 10% formalin in PBS for 5 min, rinsed with deionized water, and stained with the ALP staining kit or measured using the one-step PNPP substrate solution (Thermo Scientific, Waltham, MA, USA).

Cell viability assay
The C2C12 cells were plated on 96-well plates (three replicate plates) at the density of 2.5 × 10 3 cells/well (C2C12 cells). After the treatment with the indicated concentrations of isoflavones and soyasaponins, the cells were incubated for 3 d, and cell viability was measured using the Cell Counting Kit 8 (CCK-8) according to the manufacturer's protocol. The CCK-8 assay kit was purchased from Dojindo Molecular Technologies (Rockville, MD, USA).

Statistical analysis
All quantitative values are presented as the mean ± standard deviation. Each experiment was performed three times. Several figures show the results from one representative experiment. Statistical differences were analyzed via Student's t test and Duncan's multiple-range test using the statistical analysis software (SAS) enterprise guide 7.1 (SAS Institute Inc., Cary, NC, USA).

Isoflavone content in the germ of soybean cultivars
Isoflavone analysis in the germ of 24 Korean soybean cultivars was performed by UHPLC-DAD. Twelve isoflavones were detected in the SG (Additional file 1: Fig.  S1). The total isoflavone content ranged from 1110.9 to 3131.1 mg/100 g and the highest total isoflavone content was in the Daepung2ho cultivar, whereas the lowest one was in the Saedanback cultivar. Among isoflavones, β-glycoside (daidzin, glycitin, and genistin) and malonyl-glycoside (malonyl-daidzin, malonyl-glycitin, and malonyl-genistin) isoflavones were the major compound in SG (Table 1). The range of isoflavone content has been reported to depend on soybean cultivars [5].

Soyasaponin content in the germ of soybean cultivars
Soyasaponin analysis in the germ of 24 Korean soybean cultivars was performed by UHPLC-CAD. Only four compounds out of 10 soyasaponin standards were detected (Additional file 1: Fig. S2); the total soyasaponin contents ranged from 1173.5 to 3582.3 mg/100 g; soyasaponin Aa content ranged from 1831.6 to 2195.2 mg/100 g; soyasaponin Ab content ranged from 102.4 to 3478.1 mg/100 g; soyasaponin Ba content ranged from 23.1 to 45.4 mg/100 g, and soyasaponin Bb contents ranged from 36.3 to 136.5 mg/100 g. The highest total soyasaponin content was in the Seonpung cultivar, whereas the lowest content was in the Saedanback cultivar (Table 2). These various ranges of soyasaponin content have been reported to depend on soybean cultivars [5]. The content of soyasaponins Ab and Aa was high in the total soyasaponin content and the soyasaponin phenotype in SG was largely divided into Aa and Ab (Table 2). These results were similar to those that have been previously reported [1,2].

Isoflavone and soyasaponin in SG stimulate BMP-2-induced osteoblast differentiation in C2C12 cells
To study the effects of isoflavone and soyasaponin in SG on BMP-2-mediated osteogenesis, C2C12 cells were incubated with various concentrations of 12 isoflavones and 4 soyasaponins, followed by BMP-2 (100 ng/mL). As shown in Figs. 1a
Calibration curve for isoflavone standards analyzed by UHPLC-DAD. Figure  S4. Calibration curve for soyasaponin standards analyzed by UHPLC-CAD. Table S1. Extraction efficiency of soy germ extract in 24 soybean cultivars