## 4 Discussion
This is followed by bone remodeling and mineral metabolism [64; 65]. HR1901-W significantly increased the proliferation and ALP activity of MC3T3-E1 osteoblasts. The expression of the osteoblast differentiation factors Runx2, ALP, and OCN was significantly inhibited by HR1901-W treatment in the MC3T3-E1 osteoblasts. We also confirmed that 2-furoic acid, the chemical component identified in HR1901-W, increased osteoblast proliferation and ALP activity. When given orally to ovariectomized rats, 2-furoic acid prevented bone loss by selectivelyinhibiting bone resorption activity by osteoclasts [45]. Plant extracts may contain many individual components that elicit a biological effect [66]. The physiological activity of plant extracts may be synergistic, additive, or antagonistic due to the mixture of various compounds [67; 68; 69]. Therefore, further studies are needed on the potential of 2-furcic acid to affect the bone health efficacy of HR1901-W.  
Osteoclasts are large cells that resorb mineralized matrix by secreting acids and collagenases [70]. TARP-positive mononuclear cells differentiate into committed preosteoclasts that subsequently fuse to form multinuclear cells that eventually differentiate into mature osteoclasts [71]. Excessive osteocl activity is associated with decreased bone mass and increased fractures, both of which are characteristics of osteoporosis [72]. During bone resorption, osteoclasts attach to the mineralized bone matrix to dissolve minerals and secrete protons and proteases that digest the organic compounds of the bone [73; 74; 75]. Therefore, TRAP is widely used as a marker for osteoclast differentiation. We confirmed that HR1901-W inhibited osteoclasts from secreting TRAP in a dose-dependent manner. In summary, we demonstrated that HR1901-W promotes bone formation by upregulating the differentiation of MC3T3-E1 osteoblasts. It also inhibits the differentiation of RAW 264.7 cells, thereby inhibiting osteoclast formation. Although the present study studied factors related to the differentiation of osteoblasts and osteoclasts at the in vitro level, it is necessary to reproduce the mechanism and perform in vivo level studies in follow-up research. Taken together, HR1901-W is effective in promoting bone formation and inhibiting bone resorption, suggesting that it is a potential natural plant-based functional food for preventing osteoporosis. The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/app12178849/s1](https://www.mdpi.com/article/10.3390/app12178849/s1)]([https://www.mdpi.com/article/10.3390/app12178849/s1](https://www.mdpi.com/article/10.3390/app12178849/s1)), Figure S1: Structure of 2-furcic acid from HR1901-W (a). Chromatogram and PDA spectrum of _Benincasa hispida_ extract (HR1901-W) separated and fractionated by preparative HPLC (b). HPLC \({}^{1}\)H-NMR and \({}^{13}\)C-NMR spectra (c,d) of HR1901-W; Figure S2: Original blots of Western blot. Original blots of Figure 4; Table S1: The content of 2-furcic acid in HR1901-W.  
Conceptualization, Y.-E.C. ; methodology, J.-M.Y. ; software, Y.-E.C. ; validation, J.-M.Y. ; formal analysis, J.-M.Y. ; investigation, Y.-E.C. ; resources, J.-H.C.; data curation, Y.-E.C. ; writing--original draft preparation, Y.-E.C. ; writing--review and editing, J.-H.C.; visualization, Y.-E.C. ; supervision, J.-H.C.; project administration, J.-H.C.; funding acquisition, J.-H.C. All authors have read and agreed to the published version of the manuscript. The research was funded by Ministry of SMEs and Startups, grant number S3004427. Not applicable. The data presented in this study are available within this article. The authors declare no conflict of interest.