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

Abstract

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].

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

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

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
figure1

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

Fig. 2
figure2

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

Availability of data and materials

All data generated or analyzed during this study are included in this published article and its Additional file 1.

References

  1. 1.

    Berhow MA, Kong SB, Vermillion KE, Duval SM (2006) Complete quantification of group A and group B soyasaponins in soybeans. J Agric Food Chem 54(6):2035–2044

    CAS  PubMed  Article  Google Scholar 

  2. 2.

    Chitisankul WT, Takada Y, Takahashi Y, Ito A, Itabashi M, Varanyanond W, Kikuchi A, Ishimoto M, Tsukamoto C (2018) Saponin composition complexities in hypocotyls and cotyledons of nine soybean varieties. LWT Food Sci Technol 89:93–103

    CAS  Article  Google Scholar 

  3. 3.

    Choi CW, Choi SW, Kim HJ, Lee KS, Kim SH, Kim SL, Do SH, Seo WD (2018) Germinated soy germ with increased soyasaponin Ab improves BMP-2-induced bone formation and protects against in vivo bone loss in osteoporosis. Sci Rep 8(1):1–12

    Article  CAS  Google Scholar 

  4. 4.

    Dai J, Li Y, Zhou H, Chen J, Chen M, Xiao Z (2013) Genistein promotion of osteogenic differentiation through BMP2/SMAD5/RUNX2 signaling. Int J Biol Sci 9(10):1089–1098

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  5. 5.

    Hubert J, Berger M, Daydé J (2005) Use of a simplified HPLC − UV analysis for soyasaponin B determination: study of saponin and isoflavone variability in soybean cultivars and soy-based health food products. J Agric Food Chem 53(10):3923–3930

    CAS  PubMed  Article  Google Scholar 

  6. 6.

    Kim EH, Ro HM, Kim SL, Kim HS, Chung IM (2012) Analysis of isoflavone, phenolic, soyasapogenol, and tocopherol compounds in soybean [Glycine max (L.) Merrill] germplasms of different seed weights and origins. J Agric Food Chem 60(23):6045–6055

    CAS  PubMed  Article  Google Scholar 

  7. 7.

    Kim HJ, Choi EJ, Kim HS, Choi CW, Choi SW, Kim SL, Seo WD, Do SH (2019) Soyasaponin Ab alleviates postmenopausal obesity through browning of white adipose tissue. J Funct Foods 57:453–464

    CAS  Article  Google Scholar 

  8. 8.

    Kim SH, Yuk HJ, Ryu HW, Oh SR, Song DY, Lee KS, Park KI, Choi SW, Seo WD (2019) Biofunctional soyasaponin Bb in peanut (Arachis hypogaea L.) sprouts enhances bone morphogenetic protein-2-dependent osteogenic differentiation via activation of runt-related transcription factor 2 in C2C12 cells. Phytother Res 33(5):1490–1500

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Kim SL, Lee JE, Kwon YU, Kim WH, Jung GH, Kim DW, Lee CK, Lee YY, Kim MJ, Kim YH, Hwang TY, Chung IM (2013) Introduction and nutritional evaluation of germinated soy germ. Food Chem 136(2):491–500

    CAS  PubMed  Article  Google Scholar 

  10. 10.

    Kudou S, Tonomura M, Tsukamoto C, Uchida T, Sakabe T, Tamura N, Okubo K (1993) Isolation and structural elucidation of DDMP-conjugated soyasaponins as genuine saponins from soybean seeds. Biosci Biotechnol Biochem 57(4):546–550

    CAS  Article  Google Scholar 

  11. 11.

    Lee IA, Park YJ, Yeo HK, Han MJ, Kim DH (2010) Soyasaponin I attenuates TNBS-induced colitis in mice by inhibiting NF-κB pathway. J Agric Food Chem 58(20):10929–10934

    CAS  PubMed  Article  Google Scholar 

  12. 12.

    Lee SJ, Seguin P, Kim JJ, Moon HI, Ro HM, Kim EH, Seo SH, Kang EY, Jk Ahn, Chung IM (2010) Isoflavones in Korean soybeans differing in seed coat and cotyledon color. J Food Compost Anal 23(2):160–165

    CAS  Article  Google Scholar 

  13. 13.

    Lijie Z, Ranran F, Xiuying L, Yutang H, Bo W, Tao M (2016) Soyasaponin Bb protects rat hepatocytes from alcohol-induced oxidative stress by inducing heme oxygenase-1. Pharmacogn Mag 12(48):302–306

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  14. 14.

    Liu KS (2012) Soybeans: chemistry, technology, and utilization. Verlag, Springer, p 4

    Google Scholar 

  15. 15.

    Liu X, Chen K, Zhu L, Liu H, Ma T, Xu Q, Xie T (2018) Soyasaponin Ab protects against oxidative stress in HepG2 cells via Nrf2/HO-1/NQO1 signaling pathways. J Funct Foods 45:110–117

    CAS  Article  Google Scholar 

  16. 16.

    De La Parra C, Castillo-Pichardo L, Cruz-Collazo A, Cubano L, Redis R, Calin GA, Dharmawardhane S (2016) Soy isoflavone genistein-mediated downregulation of miR-155 contributes to the anticancer effects of genistein. Nutr Cancer 68(1):154–164

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  17. 17.

    Shiraiwa M, Kudo S, Shimoyamada M, Harada K, Okubo K (1991) Composition and structure of “group A saponin” in soybean seed. Agric Biol Chem 55(2):315–322

    CAS  Google Scholar 

  18. 18.

    Shiraiwa M, Harada K, Okubo K (1991) Composition and structure of “group B saponin” in soybean seed. Agric Biol Chem 55(4):911–917

    CAS  PubMed  Google Scholar 

  19. 19.

    Takahashi S, Hori K, Shinbo M, Hiwatashi K, Gotoh T, Yamada S (2008) Isolation of human renin inhibitor from soybean: soyasaponin I is the novel human renin inhibitor in soybean. Biosci Biotechnol Biochem 72(12):3232–3236

    CAS  PubMed  Article  Google Scholar 

  20. 20.

    Valsecchi AE, Franchi S, Panerai AE, Rossi A, Sacerdote P, Colleoni M (2011) The soy isoflavone genistein reverses oxidative and inflammatory state, neuropathic pain, neurotrophic and vasculature deficits in diabetes mouse model. Eur J Pharmacol 650(2–3):694–702

    CAS  PubMed  Article  Google Scholar 

  21. 21.

    Yue X, Abdallah AM, Xu Z (2010) Distribution of isoflavones and antioxidant activities of soybean cotyledon, coat and germ. J Food Process Preserv 34(5):795–806

    CAS  Article  Google Scholar 

  22. 22.

    Zhang T, Wang F, Xu HX, Yi L, Qin Y, Chang H, Mi MT, Zhang QY (2013) Activation of nuclear factor erythroid 2-related factor 2 and PPARγ plays a role in the genistein-mediated attenuation of oxidative stress-induced endothelial cell injury. Br J Nutr 109(2):223–235

    CAS  PubMed  Article  Google Scholar 

  23. 23.

    Zhang W, Popovich DG (2010) Group B oleanane triterpenoid extract containing soyasaponins I and III from soy flour induces apoptosis in Hep-G2 cells. J Agric Food Chem 58(9):5315–5319

    CAS  PubMed  Article  Google Scholar 

Download references

Acknowledgements

This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project title: Evaluation and identification of metabolites and database from up-land crop, Project No. PJ01348301)” Rural Development Administration, Republic of Korea.

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Contributions

KSL contributed to the writing of the manuscript and performed the majority of data analysis. SYW performed the osteoblast differentiation study. MJL, HYK, and HMH performed minor experiments and prepared raw materials. DJL and SWC contributed to the discussion of experimental results. WDS planned and led this research. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Woo Duck Seo.

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Supplementary information

Additional file 1: Figure S1.

Chemical structures and representative chromatograms of isoflavones in the germ of 24 soybean cultivars analyzed by UHPLC-DAD. (a) Chemical structures of isoflavones, (b) isoflavone standards, and (c) Daepung2ho cultivar. The number of peaks is as follows: 1, daidzin; 2, glycitin; 3, genistin; 4, 6″-O-malonyl-daidzin; 5, 6″-O-malonyl-glycitin; 6, 6″-O-acetyl-daidzin; 7, 6″-O-malonyl-genistin; 8, 6″-O-acetyl-glycitin; 9, daidzein; 10, glycitein; 11, 6″-O-acetyl-genistin; and 12, genistein. Figure S2. Chemical structures and representative chromatograms of soyasaponins in the germ of 24 soybean cultivars analyzed by UHPLC-CAD. (a) Chemical structures of soyasaponins, (b) soyasaponin standards, and (c) Taeseon cultivar in soyasaponin Aa phenotype. (d) Daepung2ho cultivar in soyasaponin Ab phenotype. The number of peaks is as follows: 1, soyasaponin Ac; 2, soyasaponin Bc`; 3, soyasaponin Bd; 4, soyasaponin Aa; 5, soyasaponin Be; 6, soyasaponin Ab; 7, soyasaponin Bc; 8, soyasaponin Ba; 9, soyasaponin Bb; 10, soyasaponin Bb`. Figure S3. 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

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Lee, KS., Woo, SY., Lee, MJ. et al. Isoflavones and soyasaponins in the germ of Korean soybean [Glycine max (L.) Merr.] cultivars and their compound-enhanced BMP-2-induced bone formation. Appl Biol Chem 63, 26 (2020). https://doi.org/10.1186/s13765-020-00508-y

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Keywords

  • Soy germ
  • Isoflavone
  • Soyasaponin
  • Osteoblast
  • BMP-2