## 4 Discussion  
In the present study, the biological effects of APG, a natural flavone with antioxidant and anti-inflammatory properties [9; 10; 28], were explored on oral osteoblasts cultured from human biopsies. APG, thereby, might be considered as a potential active phytochemical compound to develop a therapeutic strategy for clinical practice. The bioactivity and biological effects of APG are dependent on its chemical structure: the inhibition of \(\alpha\)-glucosidase and \(\alpha\)-amylase is induced by the double bonds in the two aromatic rings and hydroxyl groups on C-7 and C-4 [33]; the immunomododulatory properties are exerted by the C-4 hydroxyl group in ring B [34]; the Liver X receptor activation is induced by the hydroxyl radicals at position 5, 7 and 4\({}^{\prime}\)[35]. The aim of this study was to evaluate whether APG could be further considered as a bioactive agent capable of promoting bone regeneration for clinical applications in dentistry. In particular, APG could be beneficial to increase the osteoinductive properties in a bone graft mixed with other heterologous biomaterials. Treatment with APG (5, 10, 20 uM) did not affect the viability of hOBs, indicating that apigenin has no cytotoxicity for these cells. This result was confirmed by the images of the morphology study at 24 h, in which any morphological changes were detected in osteoblasts, which appeared typically shaped. Moreover, 5 uM APG slightly enhanced the cell metabolic activity, indicating a pro-proliferative effect at this concentration. Therefore, a proliferation study was performed at 48 h and 72 h after the hOBs were incubated with APG at concentrations of 5, 10 and 20 uM. At 48 h, APG stimulated cell proliferation at all concentrations, in a dose-dependent manner, where the response resulted inversely to the concentration and the highest and most statistically significant growth was observed in hOBs incubated with 5 uM APG compared to control cells. These results are in accordance with previous studies; indeed, APG was found to promote osteogenic differentiation in vitro and accelerate bone formation in vivo, indicating that APG may be a promising activator for bone repair [25]. APG has been reported to improve bone loss in ovariectomized rats [24]. In addition, 5 uM APG was able to stimulate cell proliferation also at 72 h, but the result was not significative. Thus, the lower concentration, 5 uM apigenin, seems to be the suitable concentration and this represents a strength to prevent potential adverse side effects related to higher-dose administration [36]. In a study with a murine model, higher doses of apigenin (100 or 200 mg/kg) caused damage to the the liver histoarchitecture, increased ALT, AST, ALP, ROS, ratio of oxidized to reduced glutathione (GSSG/GSH) and LPO [37]. A previous in vitro study reported that higher concentrations (>50 uM) of apigenin induced intracellular ROS accumulation with consequent cell death [38]. Based upon the results of cytotoxicity, morphology and proliferation study, APG at a concentration of 5 uM (5-APG) was selected for further investigations concerning the formation of the mineralized extracellular matrix. Thus, the activity of the ALP was assessed as the early phenotypic marker for mature osteoblasts, while the mineralized nodule  
Figure 6: Effects of 5 uM of APG (5-APG) on gene expression. ALP (alkaline phosphate) (**A**), OCN (osteocalcin) (**B**), COL1 (collagen 1) (**C**) gene expression was evaluated after 7 days of treatment. Results are reported as mean expression values normalized on the housekeeping \(\beta\)-actin in triplicate with error bars representing the SD (\({}^{**}p<0.001\); \({}^{***}p<0.0001\)). formation as a phenotypic marker for the later stage. The results indicated that APG (5 \(\upmu\)M) significantly increased activity of ALP at 7 days and the mineralized nodule formation at 14 days, suggesting that apigenin accelerates mineralization in osteoblasts.