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

Improvement of bone formation is necessary for successful treatment of the bone defects associated with osteoporosis. In this study, we sought to elucidate the osteogenic activity of peanut sprouts and their bioactive components. We found that peanut sprout water extract (PSWE) enhanced bone morphogenetic protein‐2‐mediated osteoblast differentiation in a dose‐dependent manner by stimulating expression of runt‐related transcription factor 2 (Runx2) via activation of AKT/MAP kinases. We identified a major component of PSWE, soyasaponin Bb, as the bioactive compound responsible for improvement of anabolic activity. Soyasaponin Bb from PSWE enhanced expression of the osteogenic transcription factor Runx2 and alkaline phosphatase. The soyasaponin Bb content depended on sprouting time of peanut, and the anabolic action of PSWE was dependent on soyasaponin Bb content. Thus, PSWE and soyasaponin Bb have the potential to protect against bone disorders, including osteoporosis.

represents a promising strategy for treatment of bone metabolic disorders such as osteoporosis. In clinical practice, treatment efforts have focused on anti-resorptive agents, but these have not been sufficiently effective. Accordingly, new strategies for inducing osteoblast differentiation are required.
Osteoblast differentiation is a pivotal event in bone formation.
Differentiation of osteoblasts from mesenchymal progenitor cells contributes to bone formation by promoting the production of extracellular matrix, which supports ossification by closely packed sheets on the bone surface (Beloti & Rosa, 2005). Osteoblast differentiation is regulated by signaling cascades and several transcriptional factors that promote mineralization and formation of bone. Runt-related transcription factor 2 (Runx2), a transcription factor, is essential for osteoblast differentiation via its ability to induce the expression of osteoblastic downstream effectors (Komori, 2011). Furthermore, Runx2 is a pivotal mediator of signaling molecules including bone morphogenetic proteins, multifunctional growth factors belonging to the transforming growth factor beta superfamily (Chen, Zhao, & Mundy, 2004;Wang et al., 2006). Accordingly, activation of Runx2 represents a therapeutic strategy for treating osteoporosis with bone defect.
Plant-derived natural products are widely used as complementary and alternative therapies for many diseases, including osteoporosis (Jiang et al., 2015). A simple and effective tool for improving biological activity is sprouting, which increases the levels of bioactive and nutritional components in seeds. For example, sprouting of peanut increases the abundance of bioactive components including resveratrol, isoflavones, and polyphenols (Kim, Park, & Lim, 2011;Wang et al., 2005). Furthermore, peanut sprouts have neuroprotective, anti-oxidative, and anti-obesity activities (Kang et al., 2010;Kang, Ha, Woo, & Kim, 2014;Lertkaeo et al., 2017). However, previous studies have not explored the effects of peanut sprout extract (PSE) on osteogenic differentiation or the molecular mechanisms underlying any such effects.
In this study, we investigated the anabolic activity of PSE and its pharmaceutical components on bone morphogenetic protein-2 (BMP-2)-mediated osteoblast differentiation and optimized the osteogenic effect of peanut sprout water extract (PSWE) by manipulating sprouting time. Investigation of the mode of action revealed that PSWE and soyasaponin Bb, a bioactive component of the extract, potentiated the osteogenic mechanism.
2 | MATERIALS AND METHODS

| Preparation of peanut sprout extract
Sinpalkwang peanut (Arachis hypogaea L.) seeds were cultivated in 2016 in the experimental field at the National Institute of Crop Science, Jeonbuk, Korea. Peanut seeds were washed, incubated in water at 20°C for 18 hr, and then germinated at 65% humidity at 25°C in the dark. After harvesting 13 days after germination, peanut sprouts were immediately washed with clean sterile water and then freeze dried at −70°C. The dried sprouts (1.0 kg) were extracted with water, prethanol, or hexane (three extractions, 10 L each) in a shaking incubator for 2 days at 40°C. The extracts were filtered and evaporated under a vacuum and subsequently freeze dried to yield 112 g of water extract (11.2%), 85 g of prethanol extract (8.5%), and 45 g of hexane extract (4.5%) as dried powder. The concentrated extract was suspended in water, prethanol, or hexane to a final concentration of 100 mg/ml. The stock solution was further diluted in phosphatebuffered saline (PBS).

| Cell culture
All experiments were performed as described previously (Choi et al., 2017) with some modifications. Mouse mesenchymal precursor C2C12 cells were obtained from the American Type Collection (Manassas, VA, USA). C2C12 cells were maintained in alpha minimum essential medium (α-MEM) containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. To differentiate C2C12 into osteoblasts, the cells were seeded and allowed to attach and grow for 1 day, after which the medium was replaced with differentiation medium (α-MEM containing 5% FBS and 100 ng/ml rhBMP-2). The medium was changed every 3 days. Osteoblastic bone formation was monitored by alkaline phosphatase (ALP) staining.

| Cell proliferation assay
C2C12 cells were plated on 96-well plates in triplicate. After treatment with PSE or soyasaponin Bb, the cells were incubated for 3 days and then cell viability was measured using the Cell Counting Kit 8 (CCK-8; Dojindo Molecular Technologies, Rockville, MD, USA).

| RNA isolation and real-time polymerase chain reaction analysis
Primers were chosen using the Primer3 online tool. Primer sets used in this study are shown in Table S1. Total RNA was extracted from C2C12 cells using Trizol reagent (Invitrogen). First stand cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). Real-time PCR was performed using Applied Biosystems Power-Up SYBR green PCR master mix (Thermo Scientific) and detected using Quantstudio®5 Real-Time PCR (Thermo Scientific). The gene encoding GAPDH was used as an internal standard. All reactions were performed in triplicate, and data were analyzed using the 2 −ΔΔCt method (Livak & Schmittgen, 2001).

| Western blotting
C2C12 cells were washed with ice-cold PBS and lysed in lysis buffer (Cell Signaling Technology) supplemented with protease inhibitors (Roche, Basel, Switzerland). After centrifugation at 15,000 × g for 15 min, the protein in the supernatant was quantified using the detergent compatible protein assay kit (Bio-Rad, Hercules, CA, USA). The quantified proteins were denatured, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 4-12% gradient gels, and transferred onto a polyvinylidene difluoride membrane using the iBlot 2 Dry Blotting System (Thermo Scientific). Blots were incubated with primary antibodies in 1% BSA overnight at 4°C and then incubated with secondary antibodies in 5% skim milk at room temperature for 2 hr. The membranes were developed using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific) and visualized on a LAS-4000 luminescent image analyzer (GE Healthcare Life Sciences, Little Chalfont, UK). Actin and GAPDH were used as loading controls.

| Ultra-high performance liquid chromatography-charged aerosol detection (UHPLC-CAD) analysis and isolation of soyasaponin Bb from PSWE
For the analysis of the main components, PSWE was dissolved at 1 mg/ml in methanol, filtered through 0.2-μm filter units, and then subjected to UHPLC-CAD analysis. Soyasaponin Bb analysis was conducted using a reverse-phase UHPLC (Dionex Ultimate 3000, Thermo Scientific) equipped with an Acclaim™ RSLC Polar Advantage II (2.2 μm, 120 Å, 2.1 × 150 mm) column. The mobile phase was 0.1% acetic acid in water (A) or 0.1% acetic acid in acetonitrile (B). Solvent flow rate was 0.7 ml/min, and the column temperature was set to 35°C. The gradient was as follows: 0-2 min, 10% B; 5 min, 20% B; 15 min, 30% B; 20 min, 30% B; 40 min, 70% B; 50 min, 100% B; 50.1 min, 10% B; held for 9.9 min before returning to the initial conditions. Following injection of 2 μl of sample, eluted soyasaponin Bb was detected using UHPLC-CAD (Corona Veo, Thermo Scientific). The soyasaponin Bb standard was purchased from ChemFaces (Wuhan, China). For isolation, the combined PSWE was evaporated under vacuum to yield a dark green gum (120 g).

| Statistical analysis
All quantitative values are presented as means ± standard deviation.
Each experiment was performed in triplicate three to five times. Several figures show results from one representative experiment. Statistical differences were analyzed using Student's t test, and a value of p < 0.05 was considered significant.  these results suggest that the osteogenic activity of PSWE arises from its ability to enhance the expression of Runx2, which is required for osteoblast differentiation. FIGURE 2 Peanut sprout water extract (PSWE) stimulates bone morphogenetic protein-2 (BMP-2)-induced expression of runtrelated transcription factor 2 (Runx2). (a) C2C12 cells were stimulated in the presence of BMP-2 (100 ng/ml) with vehicle (water) or PSWE (100 μg/ml) for the indicated times. mRNA expression levels were assessed using real-time PCR. GAPDH was used as the internal control. * p < 0.05; ** p < 0.01; *** p < 0.001 (versus vehicle control). (b) Effects of PSWE on the levels of Runx2 and alkaline phosphatase were evaluated by immune blot analysis. GAPDH was used as the internal control. One representative result from three independent experiments yielding similar results is shown FIGURE 3 Peanut sprout water extract (PSWE) induces bone morphogenetic protein-2 (BMP-2)-mediated phosphorylation ofAKT/MAP kinase signaling molecules. Following serum starvation for 1 day, C2C12 cells were pretreated with vehicle or PSWE (100 μg/ml) for 1 hr prior to BMP-2 stimulation (100 ng/ml) for the indicated times. The expression levels of the signaling molecules were evaluated by Western blotting. Actin was used as the internal control 3.4 | Identification and characterization of soyasaponin Bb, a major component of PSWE 3.6 | Soyasaponin Bb content of PSWE and its anabolic activity depend on the germination period To obtain further insight into how soyasaponin Bb enhanced osteoblast differentiation, we investigated soyasaponin Bb content and the associated effect on anabolic activity, over the course of sprouting. As shown in Figure 6a and Figure S6, the content of soyasaponin Bb increased remarkably with sprout growth time. At noncytotoxic concentrations (≤30 μg/ml; Figure 6b), PSWE significantly enhanced BMP-2-stimulated osteogenic differentiation in a manner dependent on sprouting time (Figure 6c). In addition, ALP activity was further stimulated by increasing the germination period ( Figure 6d). These results suggest that PSWE by increasing sprouting time could improve osteogenic activity by inducing soyasaponin Bb content.

| PSWE contributes to BMP
FIGURE 5 Soyasaponin Bb in peanut sprout water extract stimulates osteogenesis by inducing runt-related transcription factor 2. (a) Effect of soyasaponin Bb on bone morphogenetic protein-2 (BMP-2)-induced alkaline phosphatase (ALP) staining. Image was acquired under a light microscope (magnification, ×100). (b) ALP activity was measured. ### p < 0.001 (versus control); ** p < 0.01; *** p < 0.001 (versus BMP-2-treated group). (c) Cell viability was determined using the CCK-8 assay. (d, e) The indicated mRNA and protein expression levels were evaluated at the indicated concentrations by real-time PCR or immunoblotting. Data are expressed as fold change in mRNA level relative to control. ## p < 0.01; ### p < 0.001 (versus control); * p < 0.05; ** p < 0.01; *** p < 0.001 (versus BMP-2-treated group). Data are representative of at least three experiments [Colour figure can be viewed at wileyonlinelibrary.com] To elucidate the relationship between anabolic activity and peanut sprouting time, we examined the expression pattern of several BMP-2-dependent molecules on different sprouting days. On differentiation Day 3, the addition of BMP-2 induced the expression of osteogenic mRNAs including Runx2, ALP, and OCL, and its induction was further amplified by PSWE with increased sprouting times (Figure 7a). Furthermore, osteogenesis-related protein expression was synergistically induced by increasing the germination time (Figure 7b). These results show that PSWE made from sprouts with a longer sprouting period significantly enhanced BMP-2-mediated induction of Runx2, a transcription factor that is required for osteogenic activity.

| DISCUSSION
This study is the first to show that PSWE and soyasaponin Bb PSWE increases BMP-2-dependent osteoblast differentiation by inducing ALP expression and activity without apparent cytotoxicity.
Previous work has shown that BMP-2 promotes the commitment of the pluripotent mesenchymal precursor cell line, C2C12, into preosteoblasts capable of bone formation and mineralization. In addition, the BMP-2-induced commitment of C2C12 cells into osteoblasts leads to ALP expression, an early marker of osteoblastic differentiation.
Furthermore, we found that PSWE dramatically enhanced mRNA and protein expression of Runx2 during BMP-2-dependent osteoblast differentiation, suggesting that PSWE stimulates BMP-2dependent osteoblast differentiation by inducing osteoblast-specific transcription factors such as Runx2, which is induced during BMP-2-stimulated transdifferentiation of C2C12 cells and involved in the development of osteoblastic cells and bone formation (Wang et al., 2006). Consistent with this, PSWE treatment also induced the expression of BMP-2-induced Runx2 downstream molecules, such as ALP, OCL, and Col1a, which are known osteoblast-specific molecules (Li, Felber, Elks, Croucher, & Roehl, 2009;Lu, Robertson, & Brennan, 2004).
The effects of PSWE on osteogenic differentiation enhanced us to investigate BMP-2-related signaling pathways. BMP-2 activates AKT, MAP kinases, and Smad molecules in osteoblastic cells. Interestingly, PSWE treatment did not affect the phosphorylation of Smad, a major BMP-2-dependent signaling molecule required for osteogenesis but activated AKT and MAP kinases, which are downstream components of the Ras-PI3K signaling pathway whose activation promotes osteoblast differentiation (Ghosh-Choudhury, Mandal, & Choudhury, 2007). Furthermore, BMP-2 stimulation of AKT and MAP kinases leads to activation of Runx2 (Bokui et al., 2008;Mukherjee, Wilson, & Rotwein, 2010). Therefore, these data suggest that PSWE might enhance osteogenic differentiation by activating the AKT/MAP-Runx2 signaling pathway.
Saponins are present in hundreds of different types of plants and foods including beans, chickpeas, peanuts, quinoa, and soy. In the Fabaceae, soyasaponins can be classified into Groups A and B according to their aglycone structure, that is, the presence of Soyasapogenol A or B (Zhang & Popovich, 2009). Interestingly, we found that PSWE with osteogenic activity contained high levels of soyasaponin Bb, which has anti-oxidative, anti-carcinogenic, cardiovascular protective, and hepatoprotective effects (Gurfinkel & Rao, 2003;Jiang, Zhong, Qi, & Ma, 1993;Kinjo et al., 2003;Lee, Park, Yeo, Han, & Kim, 2010). However, its osteogenic activity has not been previously reported. In this study, we showed that soyasaponin Bb in PSWE enhanced BMP-2-dependent osteogenesis in a dose-dependent manner via induction of Runx2 and ALP, suggesting that soyasaponin Bb in PSWE could be the bioactive substance responsible for BMP-2induced osteoblast differentiation.
Since sprouting can generate bioactive compounds, thereby increasing the health-boosting biological effects of seeds (Chavan & Kadam, 1989;Kim, Jeong, Gorinstein, & Chon, 2012), we investigated soyasaponin Bb content, as well as its mode of anabolic action, as a function of sprouting period. The results of this analysis revealed that the concentration of soyasaponin Bb in PSWE increased with sprouting time. Moreover, the osteogenic activity of PSWE, including its effect on the BMP-2-mediated expression of Runx2, ALP, and OCL, was dependent on its soyasaponin Bb content. The results suggest that the specific osteogenic action of PSWE can be attributed to the presence and concentration of soyasaponin Bb.

| CONCLUSIONS
To the best of our knowledge, this study is the first to demonstrate that PSWE and its phytochemical soyasaponin Bb have anabolic potential in BMP-2-mediated osteoblast differentiation. Specifically, we found that PSWE and soyasaponin Bb were associated with induction of the MAP kinase-Runx2 signaling pathways required for osteoblast differentiation. Induction of Runx2 leads to the expression of factors required for bone formation, including ALP, OCL, and Col1a. In addition, the osteogenic activity of PSE is determined by the content of soyasaponin Bb. Although a detailed in vivo experiment and clinical study of the bone formation activity of PSWE should be carried out before it is applied to humans, the results presented here suggest that PSWE and soyasaponin Bb could be useful FIGURE 7 Peanut sprout water extract (PSWE) stimulates runtrelated transcription factor 2 and alkaline phosphatase expression as a function of the length of the germination period. (a) The effect of PSWE on bone morphogenetic protein-2 (BMP-2)-induced mRNA expression was analyzed by real-time PCR as described in Figure 2a. Glyceraldehyde 3-phosphate dehydrogenase was used as the internal control. ## p < 0.01; ### p < 0.001 (versus control); * p < 0.05; ** p < 0.01 (versus bone morphogenetic protein-2 [BMP-2]-treated group). Data are representative of at least three experiments. (b) Effects of PSWE on BMP-2-stimulated protein levels were evaluated using Western blot analysis. One representative result from three independent experiments yielding similar results is shown biosynthesis and prepare sprouts as a functional vegetable. Journal of Agricultural and Food Chemistry, 53 (2)

SUPPORTING INFORMATION
Additional supporting information may be found online in the Supporting Information section at the end of the article.