# _Benincasa hispida_ Extract Prevents Ovariectomy-Induced Osteoporosis in Female ICR Mice
## 4 Discussion  
In this study, the efficacy of BHE in improving bone health was compared using genistein, a phytoestrogen, as a positive control. As shown in Table 1, although there was no significant difference, ovariectomized mice had increased body weight and FER compared with the sham group. Estrogen is an important regulator of lipid metabolism, and decreased estrogen secretion after menopause leads to obesity [39; 40]. Weight gain in the ovariectomy model has been reported as a mechanism of action that increases subcutaneous fat and estrogen secretion from adipose tissue [41; 42]. In addition, administration of the phytoestrogen genistein has been shown to inhibit weight gain in OVX mice [43; 44]. However, in this study, no difference in body weight or FER between the experimental groups of ovariectomized mice was noted. These results are attributed to the differences between the duration and dose of genistein and BHE, and the age at ovariectomy [45; 46]. Bone remodeling is a continuous iterative process of creating and resorbing new bone to replace old bone. Osteoblasts play a leading role in the process of creating new bone, whereas osteoclasts play a leading role in the process of resorption, which destroys old bones [47]. Estrogen deficiency induces osteoclast activity and decreases bone mass, leading to osteoporosis. Estrogen deficiency induces bone loss and osteoporosis owing to increased osteoclast activity, and ovariectomy has been reported to be associated with decreased bone  
Figure 4: Effects of _Benincasa hispida_ extracts (BHE, HR1901-W) on bone formation markers in ovariectomy-induced osteoporosis mice. The concentration of (**a**) B-ALP, and (**b**) estradiol 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. BALP, bone-specific alkaline phosphatase; OVX, ovariectomy. formation [48; 49]. In agreement with data from the literature, we observed that ovariectomy induced significant reductions in femur weight, BMC, and BMD compared with the sham group (Figure 1). However, supplementation with 2-furoic acid and HBHE (200 mg/kg) inhibited the reduction in femur weight, BMC, and BMD in OVX mice. Estrogen deficiency increases RANKL, which increases osteoclast activity and decreases osteoclast apoptosis [50]. Binding of RANKL to RANK, a surface receptor of osteoclast progenitor cells, induces osteoclast maturation and bone resorption. In addition, OPG, a soluble decoy receptor produced in osteoblast lineage cells, blocks the binding between RANKL and RANK by binding to RANKL and inhibiting osteoclast formation to prevent excessive bone resorption [36]. Estrogen suppresses RANKL production in osteoblasts and T&B lymphocytes and increases OPG production in osteoblasts [51]. Estrogen deficiency has been reported to change the RANKL/OPG ratio in a direction that favors bone resorption, and ovariectomy is associated with an increase in RANKL and osteoclast numbers and a decrease in OPG production [52; 53]. In agreement with data from the literature, we observed that ovariectomy induced significant increases in osteoclast count and RANKL production, and decreased OPG production (Figures 2 and 3a-c). However, 2-furoic acid and HBHE (200 mg/kg) supplementation inhibited the elevation of RANKL and RANKL/OPG ratio and reduced osteoclast number and OPG in OVX mice. During bone resorption, osteoclasts secrete acid and proteolytic enzymes between the cell membrane and the bone matrix. Acid plays a role in dissolving hydroxyapatite, and proteolytic enzymes induce the breakdown of bone matrix proteins, such as collagen [54]. During the breakdown of bone collagen, DPD is released from the bone matrix and is used as a biomarker for bone resorption.