Therefore, the Ganoderic-acid-G question remains as to the exact role and mechanism by which bovine CP compounds impart this regulatory control on osteoblast proliferation, because the correct cell cycle regulated by a series of cell cycle control proteins is an important guarantee of cell proliferation, and bovine CP compound treatment led to increased cell cycle arrest in G2/S phase with prolonged incubation time. We concluded that bovine CP compounds can affect the Salvianolic-acid-B proliferation of MC3T3-E1 cells by promoting DNA synthesis. Furthermore, MC3T3-E1 cells were used to investigate the influence of CP compounds on the regulation of osteoblastassociated genes, namely Runx2, ALP, Col1a1, and OC. These genes are major phenotypic markers for pre-osteoblast differentiation during bone formation. During early stages of osteoblast differentiation, osteoblasts synthesize Col1a1 and other matrix proteins, followed by the production of ALP and other osteoblastic differentiation markers, ultimately leading to the induction of extracellular matrix calcification. Runx2 determines osteoblast differentiation at early stages from multipotent mesenchymal cellsand is a master regulatory gene in bone formation. Mutations in Runx2 have been identified in patients with cleidocranial dysplasia, which is a rare autosomal dominant skeletal dysplasia characterized by delayed closure of the cranial sutures, and Runx2 has been shown to regulate the expressions of type I collagen and OC. In this study, we found that both the mRNA and protein levels of Runx2 were increased in the bovine CP compound-treated cells compared with the CN cells, demonstrating that CP compounds promoted the differentiation of osteoblasts by upregulating the level of Runx2 expression. ALP is a marker of bone formation and differentiation of osteoblasts. It hydrolyzes pyrophosphate and provides inorganic phosphate to promote mineralization in osteoblasts. Our results showed that ALP activity was detected in the early stages and increased concomitantly with osteoblast differentiation. OC is the most abundant protein in the bone matrix, and is essential for hydroxyapatite binding and deposition in the extracellular matrix of bone. Our findings suggested that bovine CP compounds significantly increased the amount of OC at a late stage. Most importantly, bovine CP compounds significantly promoted the formation of mineralized nodules in MC3T3-E1 cells. Taken together, these results suggest that bovine CP compounds exerted a dominant effect on the stimulation of bone formation. Col1a1 is the most abundant protein synthesized by active osteoblasts and is essential for mineral deposition, and its expression therefore represents the start of osteoblast differentiation. As bone health supplements, bovine CP compounds are thought to stimulate increased synthesis of collagen. Mitogenactivated protein kinases and extracellular signal-regulated kinasesplay important roles in osteoblast proliferation and differentiation. Recently, it was reported that treatment with CH from porcine skin gelatin significantly increased the collagen content and expression of the Col1a1 gene through phosphorylation of ERK1/2. However, at the molecular level, we found that the expression of Col1a1 mRNA was not significantly upregulated after treatment with bovine CP compounds, which is consistent with a report that none of the collagen hydrolysates obtained from different sources stimulated the biosynthesis of collagen.