1. Introduction
Bones are dynamic tissues of various types of cells that undergo regeneration and repair processes known as bone remodeling. Osteoclasts and osteoblasts are cells that play an important role in bone maintenance [ 1 ], and the balance between osteoclastic and osteoblastic activity is important to maintenance of bone homeostasis. Increased numbers or activity of osteoclasts in bone homeostasis causes bone loss in diseases such as osteoporosis, Paget’s disease, rheumatoid arthritis, and periodontal disease [ 2 ].
Osteoporosis, which is the most common bone disease in the world, is associated with bone mass decrease and fracture risk. Patients with osteoporosis have a low bone density and weakened microstructure, which is likely to lead to pathological fractures. Fractures caused by osteoporosis are recognized as a global public health problem [ 3 ]. Such fractures can cause considerable pain and severe disability, leading to poor quality of life. The main cause of osteoporosis is related to increased osteoclast numbers and their bone resorption activity [ 3 ].
Osteoclasts are tartrate-resistant acid phosphatase-positive multinuclear cells (TRAP + -MNCs) formed by the fusion of several mononuclear precursors that are responsible for bone resorption. Osteoclasts remove old or weak bones, and are involved in maintenance of blood mineral levels [ 4 , 5 ]. Osteoclast differentiation is regulated by two cytokines, macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) [ 6 ]. M-CSF produced by osteoblasts (or immune cells) induces the expression of RANK and the survival signal of osteoclasts in osteoclast precursor cells [ 7 ]. RANKL secreted by osteoblast/activated T cells binds to the osteoclast receptor RANK and induces the activation of mitogen-activated protein kinase (MAPK), c-Fos, and NFATc1 [ 5 , 6 , 8 , 9 , 10 ]. NFATc1, the key transcription factor for osteoclast formation, regulates the expression of osteoclast differentiation and activation factors such as TRAP, cathepsin K, and dendritic cell-specific transmembrane protein (DC-STAMP) [ 11 , 12 , 13 , 14 , 15 ].
Studies of the development of fruit or grains containing functional ingredients and antioxidants have been conducted by many researchers all over the world. Anthocyanin is known to be an antioxidant with antidiabetic activity, as well as cholesterol lowering, anticancer, and anti-aging effects [ 16 , 17 , 18 , 19 ]. Recently, Boseokchal was developed as a new barley strain with a high content of anthocyanin [ 20 , 21 ]. In this study, we investigated the effects of Boseokchal extract on osteoclast differentiation and activation directly related to osteoporosis.
2. Results
2.1. BSC-W Inhibited RANKL-Mediated Osteoclast Differentiation
To examine whether BSC-W affected RANKL-mediated osteoclastogenesis, BMMs were cultured with RANKL and M-CSF in the presence of 0.1% DMSO (vehicle) or BSC-W (0, 1, 3, 10, 30 μg/mL) for 4 days. BMMs were differentiated into TRAP + -MNCs by RANKL, but BSC-W significantly reduced this differentiation ( Figure 1 A). When the number of TRAP + -MNCs with three or more nuclei was analyzed, the formation of osteoclasts was significantly decreased at a BSC-W concentration of more than 10 μg/mL ( Figure 1 B).
2.2. BSC-W Had No Cytotoxic Effect On Bmms
To determine whether inhibition of osteoclastogenesis was due to cytotoxicity by BSC-W, we conducted cytotoxicity studies in BMMs. Briefly, BMMs were cultured in 10% α-MEM treated with 0.1% DMSO (vehicle) or BSC-W (1, 3, 10, 30 μg/mL) for 3 days in the presence of 30 ng/mL M-CSF. As shown in Figure 1 C, BSC-W did not exert cytotoxicity toward BMMs at the concentrations used in this study ( Figure 1 C).
2.3. Effects of BSC-W on RANKL-Induced Gene Expression
To confirm the mechanism of inhibition activity of BSC-W in osteoclast differentiation, we analyzed the expression of c-Fos and NFATc1, transcription factors that regulates osteoclastogenesis, and the marker genes involved in osteoclast differentiation, such as TRAP, DC-STAMP, and cathepsin K. RANKL increased the level of mRNA expression of c-Fos and NFATc1, but BSC-W significantly decreased their expression level ( Figure 2 A,B). BSC-W also significantly reduced the mRNA expression level of TRAP, DC-STAMP, and cathepsin K ( Figure 2 C–E).
2.4. BSC-W Inhibited RANKL-Induced Protein Expression of NFATc1
We confirmed the effects of BSC-W on protein expression of c-Fos and NFATc1 using Western blot analysis. The protein expression of c-Fos and NFATc1 induced by RANKL was significantly decreased by BSC-W ( Figure 3 ). These results indicated that BSC-W inhibited NFATc1 expression and osteoclast formation.
2.5. Effects of BSC-W on RANKL-Mediated Bone Resorptive Activity of Osteoclasts
We investigated whether BSC-W affects bone resorption of osteoclasts. The osteoclasts made a wide pit area on the bone slice, but BSC-W drastically reduced the pit area in a dose-dependent manner ( Figure 4 ).
3. Discussion
Osteoporosis is a disease in which the quality of the bone deteriorates and fracture rate increases. In 2000, a total of 9 million osteoporotic fractures occurred, including 1.6 million pelvic fractures, 1.7 million forearm fractures, and 1.4 million vertebral fractures [ 22 ]. The increase in osteoporosis patients because of population aging has become a problem for the health and economy of the world [ 23 , 24 ]; therefore, investigation of osteoporosis is necessary for the prevention and treatment of osteoporosis.
Bone quality is greatly influenced by the balance between the activity of bone-resorbing osteoclasts and bone-forming osteoblasts. Osteoclasts, which originate from mononuclear cells/macrophages of bone marrow, are the only cells that can absorb bone [ 25 ]. These cells are produced and matured through various steps, such as differentiation and fusion, and the overall process is regulated by the RANKL-RANK signaling system [ 5 ]. Specifically, RANKL-RANK signaling is important to the formation of osteoclasts and for the treatment of pathological bone loss by these cells. After RANKL binds to the receptor, RANK, it rapidly activates MAPKs signaling molecules, such as p38, ERK, and JNK, and eventually promotes the activation of c-Fos-NFATc1 signaling, which is known to be an important transcription factor in osteoclast differentiation and activation [ 26 , 27 , 28 ].
Barley, which seems to have been cultivated in the Mesopotamia region since 7000 BC, is one of the major grains, along with rice, wheat, sorghum, and corn. Barley and wheat are believed to be the first crops grown by humans. In the previous studies, β-D-glucan, a representative dietary fiber of barley, decreased blood cholesterol levels in the body [ 29 , 30 ]. The value of barley as a healthy food is gradually increasing because of its excellent nutritional materials. The Boseokchal used in this study is an improved strain of hull-less waxy barley that contains high levels of the antioxidant anthocyanin and has therefore attracted attention as a functional food.
Here, we investigated the effects of Boseokchal’s water extract (BSC-W) on RANKL-induced osteoclastogenesis. BSC-W significantly reduced the differentiation of RANKL-treated macrophages into osteoclasts with no cytotoxicity, confirming that it inhibited osteoclast formation. Transcriptional factors of the NFAT family are known for being key molecules involved in the regulation of various biological systems, as well as in the formation of osteoclasts [ 31 , 32 , 33 , 34 ]. Therefore, we analyzed the expression levels of NFATc1 mRNA and protein by real-time PCR and western blotting, respectively, to check the influence of BSC-W against osteoclast differentiation. BSC-W significantly inhibited the expression levels of RANKL-induced NFATc1 mRNA and protein during osteoclast differentiation. In addition, mRNA expression of TRAP, DC-STAMP, and cathepsin K, which are differentiation/activation markers of osteoclasts, were significantly decreased by BSC-W. We also confirmed that the bone resorptive activity of osteoclasts was inhibited by BSC-W using a pit assay.
In previous papers, polyphenols and flavonoids, which are the major components of plant extracts, they are known as the inhibitive materials against osteoclast differentiation in osteoporosis. So it would be helpful in the treatment of osteoporosis [ 35 , 36 , 37 , 38 , 39 , 40 ]. According to the results of analyzing the contents of polyphenol and flavonoid component in BSC-W, the total polyphenol content was 8.73 ± 0.05 mg gallic acid equivalents/g and the total flavonoid content was 41.27 ± 1.46 mg quercetin equivalents/g. Therefore, it was confirmed that the inhibitive activity of BSC-W against the differentiation and resorption of osteoclasts caused by polyphenols and flavonoids.
Taken together, these results suggested that BSC-W could prevent osteoclast differentiation by inhibiting the expression of NFATc1 and the activities of osteoclastogenic-related factors during osteoclast formation and differentiation. Therefore, BSC-W could be applied as a useful functional food or therapeutic agent for bone diseases such as osteoporosis.
4. Materials and Methods
4.1. Preparation of the Boseokchal Water Extract
Preparation of Boseokchal water extract was performed as previously described [ 41 ]. Briefly, flour of Boseokchal was defatted three times with 1 L hexane for 24 h at room temperature. After filtration with Whatman No. 3, the residue was extracted three times with 1 L prethanol, then passed through a Büchner funnel lined with filter paper (Carl Roth, Karlsruhe, Germen, 111A, Ø100 mm). The residue of the prethanol extraction was subsequently extracted twice with 1 L of water for 24 h at room temperature, after which the liquid was concentrated on a rotary evaporator and lyophilized to give an extract. The total flavonoid and phenolic contents of BSC-W was analyzed by the method of previous papers [ 42 , 43 ].
4.2. Cell Culture and Osteoclast Differentiation
In vitro cell experiments were performed as previously published paper [ 44 ]. All cells were cultured at 37 °C under 5% CO 2 and medium was renewed every three days. Bone marrow cells (BMCs) were obtained from the femurs and tibiae of five week-old male ICR mice (n = 2: Raon Bio, Yongin, Korea). The protocol was approved by the Sunchon National University Institutional Animal Care and Use Committee (SCNUIACUC; Permit No. SCNU IACUC 2016-06). The BMCs from mice were cultured with 10 ng/mL macrophage colony-stimulating factor (M-CSF; Peprotech, Rocky Hill, NJ, USA) overnight in α-MEM (Thermo Fisher Scientific Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific Inc., Waltham, MA, USA) and 100 U/mL penicillin/streptomycin (10% FBS α-MEM) on two 10 cm culture dish. Non-adherent cells were isolated and cultured with M-CSF (30 ng/mL) in 10% FBS α-MEM on 10 cm Petri dishes for three days. Adhered cells were used as bone marrow derived macrophages (BMMs). We used the 96 well plate and cells were plated 1 × 10 4 cells/well. BMMs were cultured with RANKL (10 ng/mL; R&D Systems, Minneapolis, MN, USA) and M-CSF (30 ng/mL) in 10% FBS α-MEM for 4 days in the presence of vehicle or BSC-W with 0.1% DMSO as solvent.
4.3. Tartrate-Resistant Acid Phosphatase (TRAP) Staining
The cells were fixed with formaldehyde (10%) for 5 min, permeabilized with Triton X-100 (0.1%) for 10 min, and then incubated with TRAP solution (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 10 min. Red stained cells with three or more nuclei were counted as mature osteoclasts.
4.4. Cytotoxicity Assay for Extracts of BSC-W
BMMs were incubated with M-CSF (30 ng/mL) in 10% FBS α-MEM in the presence of 0.1% DMSO or BSC-W for 3 days. Cell viability was assessed using a cell counting kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, JP) according to the manufacturer’s protocols.
4.5. Real-Time PCR
Real-time PCR was performed as previously described [ 26 ]. BMMs were cultured with RANKL (10 ng/mL) and M-CSF (30 ng/mL) in 10% FBS α-MEM for 0, 1, 2, or 3 days in the presence of 0.1% DMSO or BSC-W. For real-time PCR, primer sets were designed ( Table 1 ) using the online primer3 program [ 45 ]. Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s protocols. cDNA was synthesized using a M-MLV cDNA synthesis kit (Enzynomics, Daejeon, Korea) according to the manufacturer’s protocols. QPCR was conducted using the TOPreal qPCR 2× PreMIX (BioRad, Hercules, CA, USA) in a Real-Time PCR Detection System (BioRad, Hercules, CA, USA). The mRNA levels of the genes were analyzed by the 2 −ΔΔ CT method [ 46 ]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal standard.
4.6. Western Blot
Western blotting was conducted as previously described [ 47 ]. Briefly, BMMs were incubated in the same manner as real-time PCR assays, after which cells were washed with phosphate-buffered saline (PBS) and lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, and 1% deoxycholate) containing protease inhibitors. Next, the supernatant containing proteins were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were subsequently transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Corporation, Billerica, MA, USA), after which membranes were blocked with 5% skim milk in TBST (10 mM Tris-HCl pH 7.5, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature. Blocked membranes were then incubated overnight at 4°C with a primary antibody, after which they were incubated with secondary antibody conjugated to horseradish peroxidase for 2 h at room temperature. Finally, the membranes were developed with CLARO TM Mucho (bio-D, Gwangmyeong, Korea) using the LAS-4000 luminescent image analyzer (Fuji Photo Film Co. Ltd., Tokyo, JP).
4.7. Bone Pit Formation Assay
BMMs were cultured with RANKL (10 ng/mL) and M-CSF (30 ng/mL) in 10% FBS α-MEM for 3 days and then treated with vehicle (0.1% DMSO) or BSC-W for additional 3 days on an Osteo Assay Plate (24 well plate; Corning Inc., Corning, NY, USA) at a density of 6 × 10 4 cells/well. Cells were removed with sodium hypochlorite (5%) for 5 min, after which the pit area was observed under a light microscope (magnification, 50×; Leica Microsystems, Wetzlar, Germany) and measured using the Image J software (NIH, Bethesda, MD, USA).
4.8. Statistical Analysis
All quantitative data are presented as the means ± standard deviations of three replicate experiments. The treatment groups were compared individually with the control group. Statistical differences were identified by Student’s t-tests and probability ( p ) values < 0.05 were considered significant ( p -values * <0.05, ** <0.01, and *** <0.001).
5. Conclusions
The water extract of Boseokchal (BSC-W) inhibited osteoclast differentiation by blocking the expression of NFATc1 during RANKL-induced osteoclastogenesis. It was also confirmed that the expression levels of osteoclast differentiation/activation factors, such as TRAP, DC-STAMP, and cathepsin K, decreased with the decreased expression of NFATc1 by BSC-W. Therefore, BSC-W could be a useful functional food or a therapeutic agent for treatment of bone diseases such as osteoporosis.
Acknowledgments
Thank you for supplying extracts used in osteoporosis experiments. The water extract of Boseokchal (BSC-W) was supplied by the National Institute of Crop Science.
Sample Availability: Samples of the compounds are not available from the authors.
Author Contributions
Conceived and designed the study: K.-J.K., Y.L., Y.-J.S., M.-K.L. Performed the study: K.-J.K., Y.L., S.-R.S., H.L. Analyzed the data: K.-J.K., M.-K.L., M.L. Wrote the paper: K.-J.K., Y.L., Y.-J.S. All authors read and approved the final manuscript.
Funding
This work was supported by the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ013524022018). The funder had no role in experiment and data analysis, decision to publish, or preparation of manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References
1. Goltzman D. Discoveries, drugs and skeletal disorders Nat. Rev. Drug Discov. 2002 1 784 796 10.1038/nrd916 12360256
2. Rodan G.A. Therapeutic Approaches to Bone Diseases Science 2000 289 1508 1514 10.1126/science.289.5484.1508 10968781
3. NIH Consensus Development Osteoporosis prevention, diagnosis, and therapy JAMA 2000 285 785 795
4. Vaananen H.K. Laitala-Leinonen T. Osteoclast lineage and function Arch. Biochem. Biophys. 2008 473 132 138 10.1016/j.abb.2008.03.037 18424258
5. Boyle W.J. Simonet W.S. Lacey D.L. Osteoclast differentiation and activation Nature 2003 423 337 342 10.1038/nature01658 12748652
6. Feng X. RANKing Intracellular Signaling in Osteoclasts IUBMB Life 2005 57 389 395 10.1080/15216540500137669 16012047
7. Kansara M. Teng M.W. Smyth M.J. Thomas D.M. Translational biology of osteosarcoma Nat. Rev. Cancer 2014 14 722 735 10.1038/nrc3838 25319867
8. Galibert L. The Involvement of Multiple Tumor Necrosis Factor Receptor (TNFR)-associated Factors in the Signaling Mechanisms of Receptor Activator of NF-kappa B, a Member of the TNFR Superfamily J. Boil. Chem. 1998 273 34120 34127 10.1074/jbc.273.51.34120 9852070
9. Wong B.R. Josien R. Lee S.Y. Vologodskaia M. Steinman R.M. Choi Y. The TRAF family of signal transducers mediates NF-kappaB activation by the TRANCE receptor J. Boil. Chem. 1998 273 28355 28359 10.1074/jbc.273.43.28355 9774460
10. Teitelbaum S.L. RANKing c-Jun in osteoclast development J. Clin. Investig. 2004 114 463 465 10.1172/JCI200422644 15314680 PMC503777
11. Takayanagi H. The Role of NFAT in Osteoclast Formation Ann. New York Acad. Sci. 2007 1116 227 237 10.1196/annals.1402.071 18083930
12. Takayanagi H. Osteoimmunology: Shared mechanisms and crosstalk between the immune and bone systems Nat. Rev. Immunol. 2007 7 292 304 10.1038/nri2062 17380158
13. Wagner E.F. Eferl R. Fos/AP-1 proteins in bone and the immune system Immunol. Rev. 2005 208 126 140 10.1111/j.0105-2896.2005.00332.x 16313345
14. Matsumoto M. Kogawa M. Wada S. Takayanagi H. Tsujimoto M. Katayama S. Hisatake K. Nogi Y. Essential Role of p38 Mitogen-activated Protein Kinase in Cathepsin K Gene Expression during Osteoclastogenesis through Association of NFATc1 and PU.1 J. Boil. Chem. 2004 279 45969 45979 10.1074/jbc.M408795200 15304486
15. Yagi M. Miyamoto T. Sawatani Y. Iwamoto K. Hosogane N. Fujita N. Morita K. Ninomiya K. Suzuki T. Miyamoto K. DC-STAMP is essential for cell–cell fusion in osteoclasts and foreign body giant cells J. Exp. Med. 2005 202 345 351 10.1084/jem.20050645 16061724 PMC2213087
16. Hyun J.W. Chung H.S. Cyanidin and Malvidin from Oryza sativa cv. Heugjinjubyeo mediate cytotoxicity against human monocytic leukemia cells by arrest of G(2)/M phase and induction of apoptosis J. Agric. Food Chem. 2004 52 2213 2217 10.1021/jf030370h 15080622
17. Ryu S. Han S. Park S. Kim H. Antioxidant activity of blackish purple rice Korean J. Crop. Sci. 2006 51 173 178
18. Ghosh M. Kim I.S. Lee Y.M. Hong S.M. Lee T.H. Lim J.H. Debnath T. Lim B.O. The Effects of Aronia melanocarpa ‘Viking’ Extracts in Attenuating RANKL-Induced Osteoclastic Differentiation by Inhibiting ROS Generation and c-FOS/NFATc1 Signaling Molecules 2018 23 615 10.3390/molecules23030615 PMC6017336 29518052
19. Dou C. Li J. Kang F. Cao Z. Yang X. Jiang H. Yang B. Xiang J. Xu J. Dong S. Dual Effect of Cyanidin on RANKL-Induced Differentiation and Fusion of Osteoclasts J. Cell Physiol. 2016 231 558 567 10.1002/jcp.24916 25545964
20. Choi J. Suh J. Min K. Lee M. Park J. Kim Y. Park T. Hwang J. Kim H. Baek S. A new colored naked barley cultivar” Zasoojeongchal” with waxy endosperm Korean J. Breed. Sci. 2007 39 244 245
21. Lee M. Kim Y. Park J. Kim M. Hyun J. Hull-less waxy barley (Hordeum vulgare L.) cultivar ‘Boseokchal’ with high anthocyanin content and purple lemma Korean J. Breed. Sci. 2014 46 456 462 10.9787/KJBS.2014.46.4.456
22. Wang Y. Harding S.V. Thandapilly S.J. Tosh S.M. Jones PJH, Ames NP. Barley beta-glucan reduces blood cholesterol levels via interrupting bile acid metabolism Br. J. Nutr. 2017 118 822 829 10.1017/S0007114517002835 29115200
23. Crabtree G.R. Olson E.N. NFAT signaling: Choreographing the social lives of cells Cell 2002 109 67 79 10.1016/S0092-8674(02)00699-2 11983154
24. Takayanagi H. Kim S. Koga T. Nishina H. Isshiki M. Yoshida H. Saiura A. Isobe M. Yokochi T. Inoue J.-I. Induction and Activation of the Transcription Factor NFATc1 (NFAT2) Integrate RANKL Signaling in Terminal Differentiation of Osteoclasts Dev. Cell 2002 3 889 901 10.1016/S1534-5807(02)00369-6 12479813
25. Hogan P.G. Chen L. Nardone J. Rao A. Transcriptional regulation by calcium, calcineurin, and NFAT Genes Dev. 2003 17 2205 2232 10.1101/gad.1102703 12975316
26. Asagiri M. Sato K. Usami T. Ochi S. Nishina H. Yoshida H. Morita I. Wagner E.F. Mak T.W. Serfling E. Autoamplification of NFATc1 expression determines its essential role in bone homeostasis J. Exp. Med. 2005 202 1261 1269 10.1084/jem.20051150 16275763 PMC2213228
27. Kim S.-H. Kim K.-J. Kang H.J. Son Y.-J. Choi S.-W. Lee M.-J. The Dual Role of Oat Bran Water Extract in Bone Homeostasis Through the Regulation of Osteoclastogenesis and Osteoblast Differentiation Molecules 2018 23 3119 10.3390/molecules23123119 PMC6320902 30487443
28. Kim K.-J. Yeon J.-T. Choi S.-W. Moon S.-H. Ryu B.J. Yu R. Park S.-J. Kim S.H. Son Y.-J. Decursin inhibits osteoclastogenesis by downregulating NFATc1 and blocking fusion of pre-osteoclasts Bone 2015 81 208 216 10.1016/j.bone.2015.07.023 26208796
29. Rozen S. Skaletsky H. Primer3 on the WWW for general users and for biologist programmers Methods Mol. Boil. 2000 132 365 386 10.1385/1-59259-192-2:365 10547847
30. Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method Methods 2001 25 402 408 10.1006/meth.2001.1262 11846609
31. Kim H. Kim K.J. Yeon J.T. Kim S.H. Won D.H. Choi H. Nam S.J. Son Y.J. Kang H. Placotylene A, an inhibitor of the receptor activator of nuclear factor-kappaB ligand-induced osteoclast differentiation, from a Korean sponge Placospongia sp. Mar. Drugs 2014 12 2054 2065 10.3390/md12042054 24705502 PMC4012465
32. Johnell O. Kanis J.A. An estimate of the worldwide prevalence and disability associated with osteoporotic fractures Osteoporos. Int. 2006 17 1726 1733 10.1007/s00198-006-0172-4 16983459
33. Harvey N. Dennison E. Cooper C. Osteoporosis: Impact on health and economics Nat. Rev. Rheumatol. 2010 6 99 105 10.1038/nrrheum.2009.260 20125177
34. Cauley J.A. Public Health Impact of Osteoporosis J. Gerontol. A Boil. Sci. Med. Sci. 2013 68 1243 1251 10.1093/gerona/glt093 23902935 PMC3779634
35. Oka Y. Iwai S. Amano H. Irie Y. Yatomi K. Ryu K. Yamada S. Inaqaki K. Oquchi K. Tea polyphenols inhibit rat osteoclast formation and differentiation J. Pharmacol. Sci. 2012 118 55 64 10.1254/jphs.11082FP 22186621
36. Londzin P. Siudak S. Cegieła U. Pytlik M. Janas A. Waligóra A. Folwarczna J. Phloridzin, an Apple Polyphenol, Exerted Unfavorable Effects on Bone and Muscle in an Experimental Model of Type 2 Diabetes in Rats Nutrients 2018 10 E1701 10.3390/nu10111701 30405066 PMC6267570
37. Feng Y.L. Jiang X.T. Ma F.F. Han J. Tang X.L. Resveratrol prevents osteoporosis by upregulating FoxO1 transcriptional activity Int. J. Mol. Med. 2018 41 202 212 10.3892/ijmm.2017.3208 29115382 PMC5746307
38. Wang X. Batubara I. Yamauchi K. Mitsunaga T. Identification and structure-activity relationship (SAR) of chemical constituents from Daemonorops draco (Willd.) Blume and selected commercial flavonoids on anti-osteoclastogenesis activity Fitoterapia 2019 138 104280 10.1016/j.fitote.2019.104280 31376421
39. Yao W. Zhang H. Jiang X. Mehmood K. Iqbal M. Li A. Zhang J. Wang Y. Waqas M. Shen Y. Effect of Total Flavonoids of Rhizoma drynariae on Tibial Dyschondroplasia by Regulating BMP-2 and Runx2 Expression in Chickens Front. Pharmacol. 2018 9 9 10.3389/fphar.2018.01251 30450047 PMC6224448
40. Adhikary S. Choudhary D. Ahmad N. Karvande A. Kumar A. Banala V.T. Mishra P.R. Trivedi R. Dietary flavonoid kaempferol inhibits glucocorticoid-induced bone loss by promoting osteoblast survival Nutrition 2018 53 64 76 10.1016/j.nut.2017.12.003 29655780
41. Teitelbaum S.L. Bone resorption by osteoclasts Science 2000 289 1504 1508 10.1126/science.289.5484.1504 10968780
42. Kang K.-Y. Hwang Y.-H. Lee S.-J. Kim J.-J. Nam S.-J. Yee S.-T. Verification of the antioxidant activity of a subterranean part of Suaeda japonica Makino Ind. Crop. Prod. 2017 109 836 842 10.1016/j.indcrop.2017.09.049
43. Herald T.J. Gadgil P. Tilley M. High-throughput micro plate assays for screening flavonoid content and DPPH-scavenging activity in sorghum bran and flour J. Sci. Food Agric. 2012 92 2326 2331 10.1002/jsfa.5633 22419130
44. Grigoriadis A. Wang Z. Cecchini M. Hofstetter W. Félix R. Fleisch H. Wagner E.F. c-Fos: A key regulator of osteoclast-macrophage lineage determination and bone remodeling Science 1994 266 443 448 10.1126/science.7939685 7939685
45. Mansky K.C. Sankar U. Han J. Ostrowski M.C. Microphthalmia transcription factor is a target of the p38 MAPK pathway in response to receptor activator of NF-kappa B ligand signaling J. Biol. Chem. 2002 277 11077 11083 10.1074/jbc.M111696200 11792706
46. Gingery A. Bradley E. Shaw A. Oursler M.J. Phosphatidylinositol 3-kinase coordinately activates the MEK/ERK and AKT/NFkappaB pathways to maintain osteoclast survival J. Cell Biochem. 2003 89 165 179 10.1002/jcb.10503 12682917
47. Wang Y. Harding S.V. Eck P. Thandapilly S.J. Gamel T.H. Abdel-Aal E.-S.M. Grow G.H. Tosh S.M. Jones P.J. Ames N.P. High-Molecular-Weight beta-Glucan Decreases Serum Cholesterol Differentially Based on the CYP7A1 rs3808607 Polymorphism in Mildly Hypercholesterolemic Adults J. Nutr. 2016 146 720 727 10.3945/jn.115.223206 26936139