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].

Isoflavones are divided into aglycones (daidzein, glycitein, and genistein), β-glycoside (daidzin, glycitin, and genistin), acetyl-glycoside (acetyl-daidzin, acetyl-glycitin, and acetyl-genistin), and malonyl-glycosides (malonyl-daidzin, malonyl-glycitin, and malonyl-genistin) [12]. Isoflavones are known to exhibit biological activities such as anti-oxidant, anti-cancer, anti-diabetic, and bone health [4, 16, 20, 22].

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, anti-obesity, 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, anti-inflammatory, 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 and soyasaponins on BMP-2-dependent osteoblast differentiation.

Materials and methods

Chemicals and reagents

Water, acetonitrile and methanol (HPLC grade) were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Aglycones (daidzein, glycitein, and genistein) and β-glycoside (daidzin, glycitin, and genistin) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Acetyl-glycosides (acetyl-daidzin, acetyl-glycitin, and acetyl-genistin) and malonyl-glycosides (malonyl-daidzin, malonyl-glycitin, and malonyl-genistin) were purchased from Nacalai tesque (Nijo Karasuman Nakagyo, Kyoto, Japan). Soyasaponin Aa, soyasaponin Ab, soyasaponin Ac, soyasaponin Ba, soyasaponin Bb, soyasaponin Bc, soyasaponin Bc`, soyasaponin Bd, and soyasaponin Be were purchased from ChemFaces (Wuhan, Hubei, China). Soyasaponin Bb` was purchased from ChromaDex (Irvine, CA, USA). Recombinant human bone morphogenetic protein-2 (rhBMP-2) was purchased from R&D Systems (Minneapolis, MN, USA). Penicillin, streptomycin, cell culture medium and fetal bovine serum (FBS) were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). All other chemicals and solvents used in the current study were of analytical grade.

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 AcclaimTM 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.

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 × 103 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).

Results and discussion

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].

Table 1 Isoflavone content of soy germ in 24 soybean cultivars
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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].

Table 2 Soyasaponin content of soy germ in 24 soybean cultivars
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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 and 2a, isoflavones and soyasaponins induced ALP expression in a dose-dependent manner in the presence of BMP-2. Consistent with this result, isoflavones and soyasaponins considerably enhanced the BMP-2-stimulated ALP activity in a dose-dependent manner (Figs. 1b and 2b), especially soyasaponin Ab. Isoflavones and soyasaponins did not show cytotoxicity (Figs. 1c and 2c). Our study determined that Seonpung cultivar had a higher concentration of soyasaponin Ab than that in other cultivars (Table 2). The results suggest that Seonpung cultivar is promising functional food materials for preventing and improving bone loss disorders including osteoporosis. Further research is needed to examine the soyasaponin Ab content in Seonpung cultivar according to various environmental factors because phytochemical is influenced by the environmental factors [1].

Fig. 1
figure 1

Isoflavones enhance osteoblast differentiation. a C2C12 cells were cultured for 3 d with BMP-2 (100 ng/mL) with either the vehicle (DMSO) or the indicated concentration of isoflavones. Osteoblast differentiation was visualized by ALP staining. b The ALP activity was determined by measuring absorbance at 405 nm. ###p < 0.001 (versus control); ***p < 0.001 (versus BMP-2-treated group). c The effects of isoflavones on the C2C12 cell viability were evaluated by the CCK-8 assay. The data are shown as the mean ± SD and are representative of the three experiments

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Fig. 2
figure 2

Soyasaponins enhance osteoblast differentiation. a C2C12 cells were cultured for 3 d with BMP-2 (100 ng/mL) with either the vehicle (DMSO) or the indicated concentration of soyasaponins. Osteoblast differentiation was visualized by ALP staining. b The ALP activity was determined by measuring absorbance at 405 nm. ###p < 0.001 (versus control); ***p < 0.001 (versus BMP-2-treated group). c The effects of isoflavones on the C2C12 cell viability were evaluated by the CCK-8 assay. The data are shown as the mean ± SD and are representative of the three experiments

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