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In a previous study (Jeong et al., 2003, Inhibition of Drynariae Rhizoma extracts on bone resorption mediated by processing of cathepsin K in cultured mouse osteoclasts. International Immunopharmacology 3, 1685–1697), treatment of osteoclasts-containing long bone cells with Drynariae Rhizoma (DR) extract prevented the intracellular maturation of cathepsin K and thus, it was considered that DR is a pro-drug of a potent bone resorption inhibitor. To further clarify the role of DR in ossification, we investigated the effects of DR on the proliferation and differentiation of osteoblastic cell lines in vitro. In this study, the bone effect of DR is studied. We assessed the effects of DR on osteoblastic differentiation in nontransformed osteoblastic cells (MC3T3-E1) and rat bone marrow cells. DR enhanced alkaline phosphatase (ALP) activity and mineralization in a dose- and time-dependent fashion. This stimulatory effect of the DR was observed at relatively low doses (significant at 50–150 μg/ml and maximal at 150 μg/ml). Northern blot analysis showed that the DR (100 μg/ml) increased in bone morphogenetic protein-2 as well as ALP mRNA concentrations in MC3T3-E1 cells. DR (60 μg/ml) slightly increased in type I collagen mRNA abundance throughout the culture period, whereas it markedly inhibited the gene expression of collagenase-1 between days 15 and 20 of culture. These results indicate that DR has anabolic effects on bone through the promotion of osteoblastic differentiation, suggesting that it could be used for the treatment of common metabolic bone diseases such as osteoporosis. [Copyright &y& Elsevier]

Effects of traditional Korean (Hanbang) medicine, Drynariae rhizoma (DR), on the protease activity of bone loss-initiation in rats and mice were investigated. Ethanol extracts-DR (EE-DR) and water extracts-DR (WE-DR) were identified as potent inhibitor of cathepsins K and L. The original WE-DR inhibits cathepsins K and L with IC50 values of 3.7 µg/ml and 4.5 µg/ml, respectively. EE-DR was more potent than that of WE-DR, because the inhibitions of cathepsin K and L increased to 0.5 µg/ml and 0.8 µg/ml, respectively. The FE-DR was proved to be the most potent. EE-DR was found to be a potent inhibitor of cathepsins K with a Ki value of 5.0 µg/ml for cathepsin K. The activity was increased by 10-fold when the assay is performed in the presence of glutathione at pH 7.0, which favors the formation of a GSH thiolate anion. Thus, it is suggested that this increase in potency is probably due to an enhanced chemical reactivity of the extract mixtures toward the thiolate of the active site of the enzyme. WE-DR exhibited time-dependent inhibition which allowed us to determine the association and dissociation rate constants with cathepsin K. Finally, EE-DR inhibits bone resorption in an in vitro assay involving mouse osteoclasts and bovine bone with an IC50 value of 70 µg/ml. WE-DR represents a new herbal formulation inhibiting cathepsin K and L activity and proteolysis of bone collagen. These results strongly suggest that DR is effective for preventing the development of bone loss induced by cathepsin K. This result also suggested that the DR is effective for bone resorptive action in bone cells. [ABSTRACT FROM AUTHOR]

In the traditional Korean medicine, Drynariae Rhizoma (DR) [Drynaria fortunei (kunze) J. Sm] has been reported as a good enhancer for bone healing. In this experiment, we investigate the effects of DR on bone resorption using the bone cells culture. Different concentrations of crude extract of DR were added to mouse bone cells culture. The mitochondria activity of the bone cells after exposure was determined by colorimetric MTT assay. It was demonstrated that DR has potential effects on the bone cells culture without any cytotoxicity. The most effective concentration of DR on bone cells was 100 μg/ml. On the other hand, cathepsin K (Cat K) is the major cysteine protease expressed in osteoclasts and is thought to play a key role in matrix degradation during bone resorption. In this study, Mouse long bone cells including osteoclasts and osteoblast were treated with the PI3-kinase inhibitor, wortmannin (WT), and a specific inhibitor of protein kinase C (PKC), calphostin C. Although WT prevented the osteoclast-mediated intracellular processing of Cat K, calphostin C did not. Similarly, treatment of osteoclasts-containing long bone cells with Drynariae Rhizoma (DR) extracts prevented the intracellular maturation of Cat K, suggesting that DR may disrupt the intracellular trafficking of pro Cat K. This is similar to that of WT. Since secreted proenzymes have the potential to reenter the cell via mannose-6-phosphate (M6P) receptor, to prevent this possibility, we tested WT and DR in the absence or presence of M6P. Inhibition of Cat K processing by WT or DR was observed in a dose-dependent manner. Furthermore, the addition of M6P resulted in enhanced potency of WT and DR. DR dose-dependently inhibited in vitro bone resorption with a potency similar to that observed for inhibition of Cat K processing. [Copyright &y& Elsevier]