Cissus quadrangularis enhances alkaline phosphatase activity and mineralization in osteoblasts

Cissus quadrangularis (CQ) was indicated in traditional medicine for the treatment of bone healing, though little is known about its effects on osteoblast and bone formation. Objectives: The aim of this study was to determine the effects of CQ on osteoblast differentiation and function. Methods: CQ was extracted with ethanol by means of soxhlet and characterized by Thin Layer Chromatography. Cytotoxicity test of ethanol extract of CQ (CQ-E) was performed by MTT assay in MC3T3-E1 cells (MC). Cells were then cultured in alpha MEM medium containing 50 µg/ml ascorbic acid and 2 mM β-glycerophosphate. The mRNA expressions of osteoblast-related genes in CQ-E treated MC and control were analyzed by RT-PCR. The analyses of alkaline phosphatase (ALP) activity and alizarin red S staining were performed in the presence or absence of CQ-E. Inhibition of ALP activity was carried out by addition of SB203580, a p38 Mitogen Activated Protein Kinase (MAPK) inhibitor. Statistical analyses were performed by ANOVA and Tukey's test. Results: MTT assay demonstrated that when treated with 500 and 200 µg/ml of CQ-E, viability of MC were significantly decreased as compared with control while at the concentrations of 100 and 50 µg/ml, no cytotoxic effect was observed. The mRNA expressions of Cbfa1, osterix and osteocalcin were not affected by 100 µg/ml CQ-E treatment. However, at week 3 of culture, the extent of mineralized nodules stained with alizarin red S was markedly increased in CQ-E- treated MC in comparison with control. Moreover, ALP activity was significantly increased in treated cells. The addition of SB203580 resulted in significantly decreased ALP activity induced by CQ-E. Conclusion: These results suggested that CQ may regulate matrix mineralization by enhancing ALP activity. The increased ALP activity effect of CQ is likely mediated through p38 MAPK pathway. Supported by Faculty of Dentistry Thammasat University.