Effects of Triterpene Soyasapogenol B from Arachis hypogaea (Peanut) on Differentiation, Mineralization, Autophagy, and Necroptosis in Pre-Osteoblasts.

Triterpenes are a diverse group of natural compounds found in plants. Soyasapogenol B (SoyB) from Arachis hypogaea (peanut) has various pharmacological properties. This study aimed to elucidate the pharmacological properties and mechanisms of SoyB in bone-forming cells. In the present study, 1-20 µM of SoyB showed no cell proliferation effects, whereas 30-100 µM of SoyB increased cell proliferation in MC3T3-E1 cells. Next, osteoblast differentiation was analyzed, and it was found that SoyB enhanced ALP staining and activity and bone mineralization. SoyB also induced RUNX2 expression in the nucleus with the increased phosphorylation of Smad1/5/8 and JNK2 during osteoblast differentiation. In addition, SoyB-mediated osteoblast differentiation was not associated with autophagy and necroptosis. Furthermore, SoyB increased the rate of cell migration and adhesion with the upregulation of MMP13 levels during osteoblast differentiation. The findings of this study provide new evidence that SoyB possesses biological effects in bone-forming cells and suggest a potentially beneficial role for peanut-based foods.

Soyasapogenol-B as a Potential Multitarget Therapeutic Agent for Neurodegenerative Disorders: Molecular Docking and Dynamics Study.

Neurodegenerative disorders involve various pathophysiological pathways, and finding a solution for these issues is still an uphill task for the scientific community. In the present study, a combination of molecular docking and dynamics approaches was applied to target different pathways leading to neurodegenerative disorders such as Alzheimer's disease. Initially, abrineurin natural inducers were screened using physicochemical properties and toxicity assessment. Out of five screened compounds, a pentacyclic triterpenoid, i.e., Soyasapogenol B appeared to be the most promising after molecular docking and simulation analysis. Soyasapogenol B showed low TPSA (60.69), high absorption (82.6%), no Lipinski rule violation, and no toxicity. Docking interaction analysis revealed that Soyasapogenol B bound effectively to all of the targeted proteins (AChE, BuChE MAO-A, MAO-B, GSK3ß, and NMDA), in contrast to other screened abrineurin natural inducers and inhibitors. Importantly, Soyasapogenol B bound to active site residues of the targeted proteins in a similar pattern to the native ligand inhibitor. Further, 100 ns molecular dynamics simulations analysis showed that Soyasapogenol B formed stable complexes against all of the targeted proteins. RMSD analysis showed that the Soyasapogenol B-protein complex exhibited average RMSD values of 1.94 Å, 2.11 Å, 5.07 Å, 2.56 Å, 3.83 Å and 4.07 Å. Furthermore, the RMSF analysis and secondary structure analysis also indicated the stability of the Soyasapogenol B-protein complexes.

Combinatorial biosynthesis of legume natural and rare triterpenoids in engineered yeast.

Triterpenoid saponins are a diverse group of specialized (secondary) metabolites with many biological properties. The model legume Medicago truncatula has an interesting profile of triterpenoid saponins from which sapogenins are differentiated into hemolytic and non-hemolytic types according to the position of their functional groups and hemolytic properties. Gene co-expression analysis confirmed the presence of candidate P450s whose gene expression correlated highly with that of ß-amyrin synthase (bAS). Among these, we identified CYP716A12 and CYP93E2 as key enzymes in hemolytic and non-hemolytic sapogenin biosynthetic pathways. The other candidate P450s showed no ß-amyrin oxidation activity. However, among the remaining candidate P450s, CYP72A61v2 expression highly correlated with that of CYP93E2, and CYP72A68v2 expression highly correlated with that of CYP716A12. These correlation values were higher than occurred with bAS expression. We generated yeast strains expressing bAS, CPR, CYP93E2 and CYP72A61v2, and bAS, CPR, CYP716A12 and CYP72A68v2. These transgenic yeast strains produced soyasapogenol B and gypsogenic acid, respectively. We were therefore able to identify two CYP72A subfamily enzymes: CYP72A61v2, which modifies 24-OH-ß-amyrin, and CYP72A68v2, which modifies oleanolic acid. Additionally, P450s that seemed not to work together in planta were combinatorially expressed in transgenic yeast. The yeast strains (expressing bAS, CPR, CYP72A63 and CYP93E2 or CYP716A12) produced rare triterpenoids that do not occur in M. truncatula. These results show the potential for combinatorial synthesis of diverse triterpenoid structures and enable identification of the enzymes involved in their biosynthesis.

Biosynthesis of Soyasapogenol B by Engineered Saccharomyces cerevisiae.

Soyasapogenol B is an oleanane-type pentacyclic triterpene that has various applications in food and healthcare and has a higher biological activity than soyasaponin. Saccharomyces cerevisiae is a potential platform for terpenoid production with mature genetic tools for metabolic pathway manipulation. In this study, we developed a biosynthesis method to produce soyasapogenol B. First, we expressed ß-amyrin synthase derived from Glycyrrhiza glabra in S. cerevisiae to generate ß-amyrin, as the precursor of soyasapogenol B. Several different types of promoters were then used to regulate the expression of key genes in the mevalonate pathway (MVA), and this subsequently increased the yield of ß-amyrin to 17.6 mg/L, 25-fold more than that produced in the original strain L01 (0.68 mg/L). Then, using the ß-amyrin-producing strain, we expressed soyasapogenol B synthases from Medicago truncatula (CYP93E2 and CYP72A61V2) and from G. glabra (CYP93E3 and CYP72A566). Soyasapogenol B yields were then optimized by using soyasapogenol B synthases and cytochrome P450 reductase from G. glabra. The most effective soyasapogenol B production strain was used for fermentation, and the yield of soyasapogenol B reached 2.9 mg/L in flask and 8.36 mg/L in a 5-L bioreactor with fed glucose and ethanol. This study demonstrated the heterologous synthesis of soyasapogenol B in S. cerevisiae using the combined expression of CYP93E3 and CYP72A566 in the synthesis pathway, which significantly increased the production of soyasapogenol B and provides a reference method for the biosynthesis of other triterpenes.

Soyasapogenol B Attenuates Laryngeal Carcinoma Progression through Inducing Apoptotic and Autophagic Cell Death.

Soyasapogenol B (Soy B), a constituent of soybean, has been shown to exhibit antitumor activities against different types of cancers. However, to our knowledge, no studies so far have investigated the effect of Soy B in human laryngeal carcinoma. This study, therefore, aimed to determine the effect of Soy B in human laryngeal carcinoma cell lines HeP-2 and TU212 and to elucidate the possible underlying mechanisms by which Soy B can induce its antitumor effects. The results showed that Soy B effectively attenuated the cell growth by causing G0/G1 phase cell cycle arrest in laryngeal carcinoma cell lines. Moreover, the percentage of apoptotic and autophagic cells dramatically increased upon exposure to Soy B. Western blotting results confirmed that Soy B can alter the expression levels of established markers of apoptosis and autophagy. Interestingly, both apoptosis inhibitor (ZVAD-fmk) and autophagy inhibitor (3-MA) could partially reverse the effect of Soy B, while blocking autophagy did not cause obvious alteration in the percentage of apoptotic cells. Similarly, in vivo studies validated that Soy B could effectively reduce the size of the tumor and induce apoptosis and autophagy in tumor tissues. Collectively, these results suggested that Soy B can exert anticancer activities against laryngeal carcinoma through inducing apoptotic and autophagic cell death. Our study highlighted the potential role of Soy B as a chemotherapeutic agent for laryngeal carcinoma. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1851-1858, 2020. © 2019 American Association for Anatomy.

Soyasapogenols reduce cellular triglyceride levels in 3T3-L1 mouse adipocyte cells by accelerating triglyceride lipolysis.

Soyasapogenol is a soyasaponin aglycone, which has been suggested to exert a more potent function than the glycoside form. In this study, the effect of soyasapogenol A and B on cultured adipocyte cell function was investigated using mouse 3T3-L1 adipocyte cells. 3T3-L1 cells were treated with insulin, dexamethasone, and 3-isobutyl-1-methylxanthine for differentiation to adipocytes, and the cells were then cultured in the presence of soyasapogenol A or B (6.25 or 12.5 µM). The media were harvested and refreshed every 2 d. After a 10 d culture, the cells were harvested and the triglyceride content of the cells was determined. The triglyceride content of soyasapogenol B-treated cells was significantly lower than those of vehicle-treated cells. Glycerol and free fatty acid levels in the soyasapogenol-treated cell media were higher than those in vehicle cells. However, there was no difference in the level of adipose triglyceride lipase among soyasapogenol A-, soyasapogenol B-, and vehicle-treated cells. The secreted adiponectin and resistin levels of soyasapogenol-treated cell media were also different compared with those of vehicle-treated cells. Especially, the secreted resistin level in soyasapogenol B-treated cell media was obviously reduced compared with that of vehicle-treated cells. Taken together, these results suggest that soyasapogenol B exerted an anti-obesity and anti-diabetic effect on adipocytes by lowering the cellular triglyceride level by accelerating triglyceride lipolysis with reduced resistin secretion.

Improvement of Aspergillus flavus saponin hydrolase thermal stability and productivity via immobilization on a novel carrier based on sugarcane bagasse.

Soyasapogenol B (SB) is known to have many biological activities such as hepatoprotective, anti-inflammatory, anti-mutagenic, antiviral and anticancer activities. Enzymatic conversion of soyasaponins to SB was carried out using saponin hydrolase (SH) extracted from Aspergillus flavus. The partially purified enzyme was immobilized on different carriers by physical adsorption, covalent binding or entrapment. Among the investigated carriers, Eupergit C and sugarcane bagasse (SCB) activated by DIC and NHS were the most suitable two carriers for immobilization (the immobilized forms recovered 46.5 and 37.1% of the loaded enzyme activity, respectively). Under optimized immobilization conditions, immobilized SH on Eupergit C and on activated SBC recovered 87.7 and 83.3% of its original activity, respectively. Compared to free SH, immobilized SH on Eupergit C and on activated SCB showed higher optimum pH, activation energy, half-lives and lower deactivation constant rate. Also, their SB productivities were improved by 2.3- and 2.2-folds compared to free SH (87.7 and 83.3 vs. 37.5%, respectively). Hence, being SCB more sustainable and an inexpensive material, it can be considered a good alternative to Eupergit C as a support for SH immobilization. SH immobilization on industrially applicable and inexpensive carrier is necessary to improve SB yield and reduce its production cost. The chemical structure of SCB and the resulting cellulose derivatives were studied by ATR-IR spectroscopy. The thermal analysis technique was used to study the chemical treatment of SCB and coupling with the enzyme. This technique confirmed the removal of lignin and hemicellulose by chemical treatment of SCB.

Soyasapogenol B and Genistein Attenuate Lipopolysaccharide-Induced Memory Impairment in Mice by the Modulation of NF-κB-Mediated BDNF Expression.

Lactobacillus plantarum C29-fermented defatted soybean (FDS), which contains soyasaponins such as soyasaponin I (SI) and soyasapogenol B (SB) and isoflavones such as genistin (GE) and genistein (GT), attenuated memory impairment in mice. Moreover, in the preliminary study, FDS and its soyasaponins and isoflavones significantly inhibited NF-κB activation in LPS-stimulated microglial BV2 cells. Therefore, we examined the effects of FDS and its constituents SI, SB, GT, and GE on LPS-induced memory impairment in mice. Oral administration of FDS (80 mg/kg), which has higher concentrations of SB and GE than DS, recovered LPS-impaired cognitive function in Y-maze (55.1 ± 3.5%) and passive avoidance tasks (50.9 ± 19.2 s) to 129.2% (74.1 ± 3.5%) and 114.2% (290.0 ± 22.4 s) of normal mice, respectively (P < 0.05). SB and GE (10 µM) also more potently attenuated LPS-impaired cognitive behavior than SI and GT, respectively. SB (10 mg/kg) was the most effective: treatment recovered LPS-impaired spontaneous alternation and latency time to 105.7% and 126.8% of normal control mice, respectively (P < 0.05). SB and GE significantly increased BDNF expression and CREB phosphorylation in LPS-treated mice and corticosterone-stimulated SH-SY5Y cells. Furthermore, SB and GE (10 µM) also significantly inhibited NF-κB activation in LPS-treated mice. These findings suggested that FDS and its constituent soyasaponins and isoflavones may attenuate memory impairment by the regulation of NF-κB-mediated BDNF expression.