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Drynariae Rhizoma (DR) has been demonstrated to be effective in promoting fracture healing in clinical use. In the study, we tried to predicate potential signaling pathways and active ingredients of DR via network pharmacology, uncover its regulation mechanism to improve large bone defects by in vivo and in vitro experiment. We total discovered 18 potential active ingredients such as flavonoids and 81 corresponding targets, in which mitogen-activated protein kinase (MAPK) signaling pathway has the highest correlation with bone defects in pathway and functional enrichment analysis. Therefore, we hypothesized that flavonoids in DR improve large bone defects by activating MAPK signaling pathway. Animal experiments were carried out and all rats randomly divided into TFDR low, medium, and high dosage group, model group and control group. 12 weeks after treatment, according to X-ray and Micro-CT, TFDR medium dosage group significantly promote new bone mineralization compared with other groups. The results of HE and Masson staining and in vitro ALP level of BMSC also demonstrated the formation of bone matrix and mineralization in the TFDR groups. Also, angiographic imaging suggested that flavonoids in DR promoting angiogenesis in the defect area. Consistently, TFDR significantly enhanced the expression of BMP-2, RUNX-2, VEGF, HIF-1 in large bone defect rats based on ELISA and Real-Time PCR. Overall, we not only discover the active ingredients of DR in this study, but also explained how flavonoids in DR regulating MAPK signaling pathway to improve large bone defects.

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.

Drynariae rhizoma has been used to prevent bone loss that occurs with increasing age. However, the chemical compounds in extracts that act on bone metabolism in herbal medicine are poorly understood. This study aimed to investigate and compare the extraction efficacy of polyphenolic compounds, antioxidant activity, and in vitro anti-osteoporosis properties of water extract (DR-DW) and ethanol extract (DR-EtOH) from D. rhizoma. Total phenolics and flavonoids were better extracted with 70% EtOH, and this extraction method also resulted in higher antioxidant activity and in vitro anti-osteoporosis properties in these extracts. In particular, the contents of phloroglucinol, protocatechuic acid ethyl ester, 2-amino-3,4-dimethyl-benzoic acid, 3-(3,5-dimethyl-pyrazol-1-yl)-benzoic acid, chlorogenic acid, syringic acid, trans-ferulic acid, (−)-epigallocatechin, epigallocatechin gallate, quercetin dehydrate, luteolin and emodin in DR-EtOH were higher than those in DR-DW. These results indicated that DR-EtOH could be a good source of natural herbs with anti-osteoporosis properties.