Demethylbelamcandaquinone B from Marantodes pumilum var. alata (Blume) Kuntze inhibits osteoclast differentiation in RAW264.7 cells

Objective: To investigate the bone-resorbing effect of demethylbelamcandaquinone B (Dmcq B) extracted from Marantodes pumilum var. alata on osteoclast differentiation in RAW264.7 cells. Methods: RAW264.7 macrophages were differentiated using RANKL into osteoclast-like cells. Then, they were treated with 10 mu g/mL Marantodes pumilum var. alata crude aqueous extract, 5 mu g/ mL dichloromethane fraction, and 0.6 mu g/mL Dmcq B and 0.06 mu g/ mL estradiol. Tartrate-resistant acid phosphatase 5b (TRACP 5b) as an osteoclast phenotypic marker was determined by TRACP staining and TRACP 5b colometric assay, and bone-resorbing pits were examined. The gene expression of pro-inflammatory cytokines (TNF-a and IL-6) was measured. Moreover, the protein expressions of pro-inflammatory cytokines (TNF-a and IL-6) and estrogen receptors were evaluated. Results: Marantodes pumilum var. alata crude aqueous extract and Dmcq B inhibited RANKL-stimulated osteoclast differentiation as evidenced by size reduction of giant multinucleated osteoclast cells, decreased TRACP 5b activity as well as the subsiding of resorbed pit area compared with normal control. In addition, they reduced the gene and protein expressions of TNF-a and IL-6. Marantodes pumilum var. alata, Dmcq B, and estradiol treatments increased the protein expressions of estrogen receptors alpha and beta in osteoclasts. Conclusions: Marantodes pumilum var. alata and its active compound, Dmcq B can inhibit osteoclast differentiation.