# _Benincasa hispida_ Extract Prevents Ovariectomy-Induced Osteoporosis in Female ICR Mice
## 3 Results
### Effect of BHE and 2-Furoic Acid on Bone Resorption Markers  
As shown in Figure 3, RANKL, OPG, and DPD were measured as serum markers of bone resorption. RANKL is an osteoclast-stimulating material that allows osteoclasts to perform the bone resorption process, while OPG inhibits bone resorption by blocking  
Figure 1: Effects of _Benicasa hispida_ extracts (BHE, HR1901-W) on femur bone morphologic parameters in ovariectomy-induced osteoporosis in female mice. **(a)** Normalized femur weight, **(b)** BMD, and **(c)** BMC of anesthetized mice at 8 weeks. All values are expressed as the mean \(\pm\) standard error of the mean (SEM). * \(p<0.05\), and *** \(p<0.001\) vs. sham group; # \(p<0.05\), # \(p<0.05\), and *** \(p<0.001\) vs. OVX group. BMC, bone mineral content; BMD, bone mineral density; OVX, ovariectomy; GEN, genistein; 2-FA, 2-furoic acid.  
Figure 2: Effects of _Benicasa hispida_ extracts (BHE, HR1901-W) on osteoclast in histological features of the representative metaphysis of a femur. **(a)** Giant hyper-nucleated osteoclast (arrowhead) within a histological section (H&E staining) and **(b)** number of osteoclasts. All values are expressed as the mean \(\pm\) standard error of the mean (SEM). * \(p<0.05\) vs. sham group; # \(p<0.05\) vs. OVX group.  
the maturation and activation of osteoclasts by RANKL. The RANKL/OPG ratio is an important factor in bone remodeling and directly involved in osteoporosis [36]. Therefore, an increase in the RANKL/OPG ratio may induce metabolic bone diseases, such as osteoporosis and osteoclasts due to increased osteoclastogenesis, osteoclast activity, and bone resorption. As shown in Figure 3a, the serum RANKL levels were significantly decreased in the 2-furoic acid (440.28 \(\pm\) 9.57 pg/mL) and HBHE (433.72 \(\pm\) 10.40 pg/mL) supplementation groups compared with the OVX group (512.55 \(\pm\) 8.61 pg/mL). Furthermore, as shown Figure 4b, serum OPG levels were increased in the 2-furoic acid (327.29 \(\pm\) 7.15 pg/mL) and HBHE (341.37 \(\pm\) 13.71 pg/mL) supplementation groups compared with the OVX group (263.36 \(\pm\) 23.93 pg/mL). These results induced changes in the RANKL/OPG ratio in favor of bone formation (Figure 3c). DPD is a bone resorption marker released by osteoclasts that breaks down collagen cross-linked with bone during bone resorption [37]. Moreover, as shown Figure 4d, serum DPD levels were decreased in the 2-furoic acid (14.72 \(\pm\) 1.49 ng/mL) and HBHE (12.04 \(\pm\) 0.30 ng/mL) supplementation groups compared with the OVX group (16.77 \(\pm\) 1.25 ng/mL). These results suggest that 2-furoic acid and HBHE induce a reduction in bone resorption markers in ovariectomy-induced osteoporosis mice.  
Figure 3: Effects of _Benincasa hispida_ extracts (BHE, HR1901-W) on bone resorption markers in ovariectomy-induced osteoporosis mice. The concentration of (**a**) RANKL, (**b**) OPG, (**c**) RANKL/OPG ratio, and (**d**) DPD was determined using enzyme-linked immunosorbent assay (ELISA) kits. All values are expressed as the mean \(\pm\) standard error of the mean (SEM). \({}^{***}\)\(p\) < 0.001 vs. sham group; # \(p\) < 0.05, ## \(p\) < 0.01, and ## \(p\) < 0.001 vs. OVX group. RANKL, receptor activator of nuclear factor kappa-B ligand; OPG, osteoprotegerin; DPD, deoxypyridinoline; OVX, ovariectomy.