## 4 Discussion  
Osteoporosis is a complex disorder triggered by several pathways. Even though the individual target studies are well documented, the investigation on phytochemicals toward multiple osteocl targets has not been yet elucidated. In this present study, we first studied the herbal substances that reported osteoprotective activities from previous studies [27, 28] with multitarget activity on osteoclasts by performing molecular docking and cluster analysis. Defining the potential target is the critical issue for drug discovery nowadays [51]. Therefore, we selected the promising multiple osteocl targets [52] in advance, including Cathepsin K, V-ATPase, and \(\alpha_{\mathrm{V}}\beta_{3}\) integrin receptor to explore the multitarget compounds using the target-based virtual screening. Network analysis provides a useful tool to study the complexity of diseases. Here, we reported an approach to study the PPI network based on the gene expressed in targeted proteins of osteoclasts. The result we presented here indicates that proteins, such as Cathepsin K, \(\alpha_{\mathrm{V}}\beta_{3}\) integrin receptor, and V-ATPase, share common characteristics and direct interactions. We then hypothesized that the inhibition of these proteins might promote the synergistic effect in the reduction in osteoclast function. We further performed the virtual screening of herbal substances toward three osteocl targets. However, the limitation of molecular docking is due to the low accuracy of the scoring function [53]. Thus, we performed a predictive model for the selection of the candidates, utilizing a combination of molecular docking and clustering approaches. The use of the clustering algorithm in filtering the virtual screening in the preliminary study  
Figure 8: The 2D and 3D binding poses of candidates in a binding site of integrin. Ligands (green) are bound with amino acids in chain A (white) and chain B (pink). The pharmacophore features are represented as green arrows (HBD), red arrows (HBA), and yellow spheres (hydrophobic property). helps to reduce the number of interested compounds with an unbiased algorithm. Several studies demonstrated the advantages of utilizing the clustering method as an additional method that efficiently helps in identifying the active compounds [54; 55]. Our study used both k-means and hierarchical clustering methods. These methods allow to find the reliable cluster and define the phytochemical candidates. As highlighted in our results, we defined four compounds, namely, rutin, sagittatoside A, icarin, and kaempferrin, obtained the multitarget activity against osteocl receptors. Our finding is in agreement with the previous experiments that showed that rutin exhibited an osteoprotective effect by improving the bone mineral density (BMD) and bone strength in the ovariectomized (OVX) rat [56; 57]. Additionally, rutin reduced the levels of acid phosphatases secreted during bone resorption in osteoclasts and increased the osteoclast activity, a biomarker enzyme for bone formation found in osteoblasts [57]. Sagittatoside A and icarin have been reported as the bioactive compounds from _Herba epimedii_ extract. The herb extract has gained more interest in various studies due to its beneficial activities, such as anti-oxidative, anti-inflammatory, and anti-tumor properties [58; 59]. This herb has been used for a decade to treat the cardiovascular disease, impotence, neurological disorder, and osteoporosis [60]. Given that _Epimedii_ extract to the OVX rats exhibited significantly increased bone strength via potent inhibiting the alkaline and acid phosphatase suggesting that the extract of _Epimedii_ helps to improve postmenopausal osteoporosis [61]. Icarin is a prenylated flavonoid glycoside that presents as a main bioactive compound retrieved form _Epimedium_ extract.