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This experiment was designed to opti‐ mize the extraction process of total flavonoids from Drynariae Rhizoma (DR) in ultrasoundassisted PEG400- (NH4)2SO4 aqueous two-phase system and to evaluate its antioxidant activity. Based on the single-factor approach, the optimal extraction process of total flavonoids from DR was optimized by response surface methodology with (NH4)2SO4 mass fraction, PEG400 mass fraction, ultrasonic power and ultrasonic time as independent vari‐ ables and the yield of total flavonoids from DR as dependent variables. The scavenging ability of DPPH· and ·OH and the iron reducing power of total flavonoids from DR were determined by using Vitamin C as a positive control. The results showed that the optimal extraction conditions for total flavonoids from DR were PEG400 mass fraction 19%, (NH4)2SO4 mass fraction 19%, ultrasonic power 180 W, and ultra‐ sonic time 40 min. The yield of total flavonoids from DR under this process was 182.98 mg/g, which was similar to the theoretical prediction. The analysis of antioxidant activity showed that the scavenging abil‐ ity of DPPH·, ·OH and iron reducing power of total flavonoids from DR increased and stabilized with the increase of concentration in a certain concentration range. The PEG400- (NH4)2SO4 aqueous twophase system was used for the extraction of total flavonoids from DR, and the optimal process conditions were reliable. [ABSTRACT FROM AUTHOR]

Drynariae Rhizoma (DR) is a functional food and traditional medicine that has been widely used for bone and joint disorders for thousands of years. In this study, 14 compounds were isolated from DR, and their structures were identified using UPLC/QTOF–MS, UPLC–ESI/LTQ–Orbitrap–HRMS, and 2D NMR and compared with those obtained in previous studies. An HPLC–PDA multi-component simultaneous quantitative determination method was developed for 12 of the 14 DR-derived compounds, excluding compounds with a content <1.5 mg. The developed HPLC method was validated based on linearity (r2 ≥ 0.999), limit of detection (0.01–0.65 μg/mL), limit of quantification (0.04–1.97 μg/mL), intra-day precision and accuracy ranges (0.06–2.85% and 95.03–104.75%, respectively), and inter-day precision and accuracy ranges (0.24–2.83% and 95.75–105.75%, respectively). The developed analysis method improved the resolution of compounds 4 and 5. In addition, this is the first quantitative analysis of compounds 7, 8, and 11 and the first simultaneous quantitative analysis of 12 compounds, including compounds 4, 7, 8, 10, 11, and 14. This study developed a rapid, accurate, and economical HPLC method for performing the simultaneous quantitative analysis of 12 secondary metabolites isolated from DR. [ABSTRACT FROM AUTHOR]

Purpose: To evaluate the anti-inflammatory properties of Drynariae rhizome (Polypodiaceae) extract (DRE) on lipopolysaccharide RAW264.7 macrophage cells and its protective effects on interleukin (IL)-1β-stimulated SW1353 chondrocyte cells. Methods: The anti-inflammatory effect of DRE (12.5, 25, 50, and 100 μg/mL) were measured by 5-lipooxygenase (LO) and cyclooxygenase (COX)-2 enzyme inhibitory activities, nitric oxide (NO) production in LPS-stimulated RAW264.7 cells using Griess reagent assay. Prostaglandins (PGE2) and leukotriene (LTB4) production were measured using enzyme-linked immunosorbent assay (ELISA). To investigate the protective effect on chondrocytes, the level of matrix metalloproteinases (MMP)-3, -9, and tissue inhibitor of matrix metalloproteinase (TIMP)-1 was determined using zymography assay and ELISA kit in SW1353 cells stimulated with IL-1β. Results: Drynariae rhizome (Polypodiaceae) extract (DRE) had a significant (p < 0.05) inhibitory effect on 5-LO and COX-2 enzymes in a concentration-dependent manner compared to untreated group. In RAW264.7 cells, levels of NO, PGE2 and LTB4 of DRE group were significantly (p < 0.05) decreased in study group (treated with DRE) at 12.5 μg/mL compared to the group treated with LPS (100 ng/mL) alone. Additionally, in SW1353 human chondrocytes stimulated with IL-1β, MMP-3 and MMP-9 were significantly (p < 0.05) suppressed in DRE-treated group compared to IL-1β group alone, with no significant impact on TIMP-1 production. Conclusion: Drynariae rhizome (Polypodiaceae) extract (DRE) possesses anti-inflammatory and cartilage protection effects. It therefore has the potential to be developed as an effective natural pharmaceutical agent for inflammation or osteoarthritis. [ABSTRACT FROM AUTHOR]

Objective Based on network pharmacology and bioinformatics, the molecular mechanism of drynariae rhizoma epimedii folium in the treatment of osteoporosis was discussed, in order to provide a new target for the treatment of osteoporosis. Methods The active ingredients of drynariae rhizoma-epimedii folium were screened through TCMSP database. The regulatory target was predicted through the UniProt database. The target for the treatment of osteoporosis was predicted according to the GEO database. The effective targets for the treatment of osteoporosis were obtained and the drug-component-target-pathway network was established using R language. The protein interaction network was constructed and the molecular docking between key active ingredients and key targets was verified using the Cytoscape software. Finally, the GO analysis and the KEGG pathway analysis of intersection genes were performed by using the DAVID database. Results Thirty-nine active ingredients and 17 therapeutic targets were obtained. The core targets in the protein interaction network were ESRI, PRKDC, HSPA8, EP300, and HSP90AA1. DAVID enrichment analysis showed that the biological process mainly covered xenobiotic metabolic process, steroid metabolic process. regulation of osteoclast differentiation. Signal pathway mainly included NF-kB signaling pathway, PI3K-AKT signaling pathway. and HIF-1 signaling pathway. Conclusion Analysis of the treatment of osteoporosis with drynariae rhizoma-epimedii folium through pharmacology and bioinformatics network enables us not only to identify the currently known biological processes and signaling pathways, but also to provide a reference for further research on the substance basis and action targets of drynariae rhizoma-epimedii folium in the treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

Drynariae Rhizoma is a kidney-tonifying herb that has a long history in clinical practice for the treatment of bone fractures and joint diseases in China. Flavonoids are considered to be its major active ingredients and are reported to ease bone loss in ovariectomized rats. However, the beneficial effects of the total flavonoids of Drynariae Rhizoma on osteoporosis caused by microgravity or mechanical inactivity remain unknown. This study assessed the effects of total Drynariae Rhizoma flavonoids (DRTF, Qihuang, Beijing, China, national medicine permit No. Z20030007, number of production: 04080081, content of DRTF _80%) against bone loss induced by simulated microgravity. A hindlimb unloading tail-suspended rat model was established to determine the effect of DRTF on bone mineral density (BMD), biomechanical strength and trabecular bone microarchitecture. Twenty-eight male Sprague-Dawley rats were divided into four groups: the baseline, control, hindlimb unloading with vehicle (HLU), and hindlimb unloading treated with DRTF (HLU-DRTF, 75 mg/kg/day) groups. Oral DRTF was administered for 4 weeks. The underlying mechanisms of the DRTF actions on disuse-induced osteoporosis are discussed. The results showed that DRTF treatment significantly increased the BMD and mechanical strength of tail-suspended rats. Enhanced bone turnover markers with HLU treatment were attenuated by DRTF administration. Deterioration of trabecular bone induced by HLU was prevented through elevated bone volume/tissue volume (BV/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th) and decreased trabecular separation (Tb. Sp). The present study provides the first evidence that DRTF prevents bone loss induced by HLU treatment, indicating its potential application in the treatment of disuse-induced osteoporosis. [ABSTRACT FROM AUTHOR]

Abstract: Drynariae Rhizoma (DR), a traditional Korea medicine, which is known for its effect to strengthen myoskeletal systems, frequently appears as the main ingredient in prescriptions for bone injuries. However, it is unclear how it pharmacologically contributes to the reformation of bone. In this study, the effect of DR on bone cells was investigated in vitro for the first time. The human osteoprecursor cells (OPC-1) were incubated in the medium with different concentrations of DR and the cell proliferation was studied. When the concentration of DR was ≤120μgml-1, the proliferation of OPC-1 was enhanced. However, the proliferation of OPC-1 was inhibited by DR with the concentrations of >250μgml-1. Under most treatments, the cells presented very pale expression for cyclooxygenase-2 (Cox-2) protein; slightly intensified band showed at the highest DR concentration, 120μgml-1 during the course of culture. On the other hand, we investigated the immunomodulatory activity of DR on cellular and humoral immunity. When different doses of ethanolic and water extracts of DR was administerd to mice, it was dose-dependently potentiated the delayed-type hypersensitivity reaction induced by both sheep red blood cells (SRBC) and oxazolone. It significantly enhanced the production of circulating antibody titre in mice in response to SRBC. But, DR did not any effect on macrophage phagocytosis. Prolonged administration of DR significantly ameliorated the total white blood cell count and also restored the immunosuppressive effects induced by cyclophosphamide. The present investigation reveals that DR possesses immunomodulatory activity. From the results, it was concluded that DR directly stimulated the proliferation, alkaline phosphatase activity, protein secretion and particularly type I collagen synthesis of OPC-1 at dose-dependent manner, and stimulated both the cellular and the humoral immunity. [Copyright &y& Elsevier]

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]