Exploration of photoprotective and antibiotic activity of wild Polypodiaceae ferns from Costa Rica.

Skin disorders affect millions of people all over the world. There are limited options to treat dermal illnesses such as vitiligo, psoriasis, and atopic dermatitis (eczema). Central American ferns are a potential source of bioactive metabolites against those diseases. Currently, Polypodium leucotomos Poir. is the only one being commercially utilized for this purpose. In this work, we evaluated the concentration of the skin bioactive compounds: quinic and chlorogenic acid, in the extract of 20 wild ferns from Costa Rica. We also evaluated the antimicrobial capabilities of the crude extracts of wild ferns and the sun protection factor (SPF) of the extracts. We found 19 out of 20 have either an important concentration of the compounds mentioned above or antimicrobial properties. Also, most samples result in higher SPF than P. aureum's rhizome. We also have studied the fern acclimatization, at different shading conditions, finding a significant influence of the culturing conditions on metabolite production. After acclimatization. So far, we demonstrate that various ferns included in this study are a potential source of treatments for skin conditions.

Active components and mechanisms of total flavonoids from Rhizoma Drynariae in enhancing cranial bone regeneration: An investigation employing serum pharmacochemistry and network pharmacology approaches.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhizoma Drynariae, as the dried rhizome of Drynaria fortunei (Kunze ex Mett.) J. Sm., is a traditional Chinese medicine for treating the injury and bone broken of falling and beating. Total flavonoids is considered as the major and effective compounds for the therapeutic efficacy of Rhizoma Drynariae. AIM OF THE STUDY: To explore the effect of total flavonoids from Rhizoma Drynariae (TFRD) on bone regeneration and the underlying mechanisms. MATERIALS AND METHODS: The effect of TFRD in various doses on bone reconstruction in cranial bone defect rats was explored in vivo. The active ingredients in TFRD-medicated serum were characterized by serum pharmacochemistry and integrated by network pharmacology analysis and target prediction. To elucidate the underlying mechanism of TFRD on bone regeneration, experimental validation in vitro was executed to assess the influence of different concentrations of TFRD-medicated serum on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). RESULTS: Micro-CT, histological examination, immunohistochemical analysis, and ELSA demonstrated that administration of TFRD could promote bone reconstruction in a rat cranial defect model. We identified 27 active components of TFRD using ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Results from CCK8, ALP, and Alizarin Red S staining revealed that TFRD-medicated serum notably enhanced BMSCs proliferation and osteogenic differentiation. qRT-PCR and Western blot harvested results consistent with those predicted by network pharmacology, providing further evidence that TFRD activated the TGF-ß signaling pathway to benefit bone regeneration. CONCLUSION: The active components of TFRD modulate the TGF-ß signaling pathway to facilitate osteogenesis, thereby repairing cranial bone defects.

Drynaria Naringin alleviated mechanical stress deficiency-caused bone loss deterioration via Rspo1/Lgr4-mediated Wnt/ß-catenin signalling pathway.

Osteoporosis is a metabolic condition distinguished by the degradation of bone microstructure and mechanical characteristics. Traditional Chinese medicine (TCM) has been employed in China for the treatment of various illnesses. Naringin, an ingredient found in Drynariae TCM, is known to have a significant impact on bone metabolism. For this research, we studied the precise potential effect of Drynaria Naringin on protecting against bone loss caused by stress deficiency. In this study, a tail-suspension (TS) test was performed to establish a mouse model with hind leg bone loss. Some mice received subcutaneous injections of Drynaria Naringin for 30 d. Trabecular bone microarchitecture was evaluated using micro-computed tomography analysis and bone histological analysis. Bone formation and resorption markers were quantified in blood samples from mice or in the supernatant of MC3T3-E1 cells by ELISA analysis, Western blotting, and PCR. Immunofluorescence was utilized to visualize the location of ß-catenin. Additionally, siRNA was employed to knockdown-specific genes in the cells. Our findings highlight the efficacy of Drynaria Naringin in protecting against the deterioration of bone loss and promoting bone formation and Rspo1 expression in a mouse model following the TS test. Specifically, in vitro experiments also indicated that Drynaria Naringin may promote osteogenesis through the Wnt/ß-catenin signalling pathway. Moreover, our results suggest that Drynaria Naringin upregulates the expression of Rspo1/Lgr4, leading to the promotion of osteogenesis via the Wnt/ß-catenin signalling pathway. Therefore, Drynaria Naringin holds potential as a therapeutic medication for osteoporosis. Drynaria Naringin alleviates bone loss deterioration caused by mechanical stress deficiency through the Rspo1/Lgr4-mediated Wnt/ß-catenin signalling pathway.

Metabolite profiling of Drynariae Rhizoma using 1H NMR and HPLC coupled with multivariate statistical analysis.

Drynariae Rhizoma has been used to treat bone diseases and kidney deficiency in traditional medicine. Recently its aqueous extract was reported to enhance memory function. Although the Japanese standards for non-Pharmacopoeial crude drugs 2022 prescribed Drynaria roosii as the botanical origin, some counterfeits and both raw and stir-fired crude drugs are available in markets. To distinguish Drynariae Rhizoma derived from D. roosii appropriately from others and verify the validity of uses of stir-fried ones, 1H NMR-based metabolite profiling coupled with HPLC were performed. Raw samples derived from D. roosii contained naringin (1), neoeriocitrin (2), 5,7-dihydroxychromone-7-O-neohesperidoside (3), caffeic acid 4-O-ß-D-glucoside (4), protocatechuic acid (5), trans-p-coumaric acid 4-O-ß-D-glucoside (6), and kaempferol 3-O-α-L-rhamnoside 7-O-ß-D-glucoside (8). Stir-fried samples were characterized by presence of 5-hydroxymethyl-2-furaldehyde (13), and were divided into two types; one possessing similar composition to raw samples (Type I) and another without above components except 5 (Type II). Quantitative analyses using qHNMR and HPLC, followed by principal component analysis demonstrated that the raw samples had higher contents of 1 (0.93-9.86 mg/g), 2 (0.74-7.59 mg/g), 3 (0.05-2.48 mg/g), 4 (0.27-2.51 mg/g), 6 (0.14-1.26 mg/g), and 8 (0.04-0.52 mg/g), and Type II had a higher content of 5 (0.84-1.32 mg/g). The counterfeit samples derived from Araiostegia divaricata var. formosana were characterized by higher content of ( -)-epicatechin 3-O-ß-D-allopyranoside (10) (1.44-11.49 mg/g) without 1 and 2. These results suggested that Drynariae Rhizoma samples derived from other botanical origins and Type II stir-fried samples cannot substitute for D. roosii rhizome.

Comparative Transcriptomic Analysis of Genes in the 20-Hydroxyecdysone Biosynthesis in the Fern Microsorum scolopendria towards Challenges with Foliar Application of Chitosan.

Microsorum scolopendria is an important medicinal plant that belongs to the Polypodiaceae family. In this study, we analyzed the effects of foliar spraying of chitosan on growth promotion and 20-hydroxyecdysone (20E) production in M. scolopendria. Treatment with chitosan at a concentration of 50 mg/L in both young and mature sterile fronds induced the highest increase in the amount of accumulated 20E. Using RNA sequencing, we identified 3552 differentially expressed genes (DEGs) in response to chitosan treatment. The identified DEGs were associated with 236 metabolic pathways. We identified several DEGs involved in the terpenoid and steroid biosynthetic pathways that might be associated with secondary metabolite 20E biosynthesis. Eight upregulated genes involved in cholesterol and phytosterol biosynthetic pathway, five upregulated genes related to the methylerythritol 4-phosphate (MEP) and mevalonate (MVA) pathways, and several DEGs that are members of cytochrome P450s and ABC transporters were identified. Quantitative real-time RT-PCR confirmed the results of RNA-sequencing. Taken together, we showed that chitosan treatment increased plant dry weight and 20E accumulation in M. scolopendria. RNA-sequencing and DEG analyses revealed key enzymes that might be related to the production of the secondary metabolite 20E in M. scolopendria.

Prediction of Rhizoma Drynariae Targets in the Treatment of Osteonecrosis of the Femoral Head based on Network Pharmacology and Experimental Verification.

BACKGROUND: Rhizoma drynariae, a classic prescription in traditional Chinese medicine, has long been used for the treatment of osteonecrosis of the femoral head (ONFH), but its potential targets and molecular mechanisms remain to be further explored. OBJECTIVE: This study aims to explore the mechanism of Rhizoma drynariae in ONFH treatment via network pharmacology and in vitro experiments. METHODS: Targets of Rhizoma drynariae and ONFH were predicted using relevant databases, and intersection analysis was conducted to screen for shared targets. A PPI network of the shared targets was built using STRING to identify the key targets. Functional enrichment analyses of Gene Ontology and KEGG pathway data were carried out using R software. The compound-target-pathway network was constructed for Rhizoma Drynariae in the treatment with ONFH using Cytoscape 3.9.0. Cell proliferation was assessed using CCK8 and apoptosis was detected using (Propidium Iodide) PI staining and western blotting. RESULTS: This study depicts the interrelationship of the bioactive compounds of Rhizoma drynariae with ONFH-associated signaling pathways and target receptors and is a potential reagent for ONFH treatment. CONCLUSION: Based on a network pharmacology analysis and in vitro experiment, we predicted and validated the active compounds and potential targets of Rhizoma drynariae, provide valuable evidence of Rhizoma Drynariae in future ONFH treatment.

Network Pharmacology-Based Dissection of the Mechanism of Drynariae Rhizoma for Low Back Pain.

Objective: To explain the potential mechanisms of Drynariae Rhizoma (DR) in the treatment of low back pain (LBP). Design: Network pharmacology was used to reveal the potential mechanisms including collecting the active ingredients of DR, analyzing the common gene targets of LBP and DR, constructing protein-protein interaction (PPI) network, collecting protein classification, performing Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and verifying significant gene targets. Results: 234 different gene targets and 18 active compounds altogether were obtained. AKT1, VEGFA, and HIF1A were deemed to be major gene targets based on the degree values. According to GO analysis, steroid metabolic process involved 42 (18.10%) potential therapeutic LBP targets, neuronal cell body involved 24 (10.30%) potential therapeutic LBP targets, and protein serine/threonine kinase activity involved 28 (12.02%) potential therapeutic LBP targets in biological process (BP), cellular component (CC), and molecular function (MF), respectively. According to KEGG and pathway interaction analyses, the PI3K-Akt signaling pathway involved 34 (15.89%) potential therapeutic LBP targets, and PI3K-Akt signaling pathway played a significant role in the treatment of LBP. The mRNA expression levels of AKT1 and HIF1A were upregulated in healthy nucleus pulposus (NP) tissue than in degenerative NP tissue. In contrast, the mRNA expression level of VEGFA was downregulated in healthy NP tissue than in degenerative NP tissue. Conclusions: In this study, we identified a potential relationship between LBP and DR in this work, as well as a synergistic mechanism of DR in the treatment of LBP, which serves as a benchmark for further in vivo and in vitro research.

Chemistry and Bioactivity of Microsorum scolopendria (Polypodiaceae): Antioxidant Effects on an Epithelial Damage Model.

Microsorum scolopendia (MS), which grows on the Chilean island of Rapa Nui, is a medicinal fern used to treat several diseases. Despite being widely used, this fern has not been deeply investigated. The aim of this study was to perform a characterization of the polyphenolic and flavonoid identity, radical scavenging, antimicrobial, and anti-inflammatory properties of MS rhizome and leaf extracts (RAE and HAE). The compound identity was analyzed through the reversed-phase high-performance liquid chromatography (RP-HPLC) method coupled with mass spectrometry. The radical scavenging and anti-inflammatory activities were evaluated for DPPH, ORAC, ROS formation, and COX inhibition activity assay. The antimicrobial properties were evaluated using an infection model on Human Dermal Fibroblast adult (HDFa) cell lines incubated with Staphylococcus aureus and Staphylococcus epidermidis. The most abundant compounds were phenolic acids between 46% to 57% in rhizome and leaf extracts, respectively; followed by flavonoids such as protocatechic acid 4-O-glucoside, cirsimaritin, and isoxanthohumol, among others. MS extract inhibited and disaggregated the biofilm bacterial formed and showed an anti-inflammatory selective property against COX-2 enzyme. RAE generated a 64% reduction of ROS formation in the presence of S. aureus and 87.35% less ROS in the presence of S. epidermidis on HDFa cells. MS has great therapeutic potential and possesses several biological properties that should be evaluated.

Pyrrosia petiolosa Extract Ameliorates Ethylene Glycol-Induced Urolithiasis in Rats by Inhibiting Oxidative Stress and Inflammatory Response.

Objective: To study the therapeutic effect and mechanism of Pyrrosia petiolosa (P. petiolosa) extract on ethylene glycol- (EG-) induced urolithiasis in rats. Methods: Thirty SD male rats were randomly divided into five groups (n = 6): control group, EG group, and P. petiolosa group (25 mg/kg, 50 mg/kg group, and 100 mg/kg). Biochemical testing was adopted for measuring the blood and urine parameters, as well as the level of superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde acid (MDA) in kidney tissues. HE staining and ELISA were utilized to observe the histopathological changes and detect the level of IL-1ß, IL-6, MCP-1, and TNF-α in the kidney tissue, respectively. And western blot was performed for checking NOX2, NOX4, TGF-ß1, p-Smad3, Smad3, p-Smad2, and Smad2 protein expression level in kidney tissues. Results: EG could significantly increase the level of blood urea nitrogen, creatinine, and Na in serum and 24-hour urinary protein, oxalate, uric acid, creatinine, calcium, and phosphorus in urine and decreased the urine volume in rats. Whereas P. petiolosa extract was able to greatly decrease the level of related parameters in serum and urine in a dose-dependent manner, but did not affect the urine pH. In addition, P. petiolosa extract notably ameliorated EG-induced renal tissue injury. Compared with the EG group, P. petiolosa extract markedly raised the level of SOD and GSH and decreased the MDA level and the expression of NOX2 and NOX4 in the kidney tissue. Moreover, P. petiolosa extract also lowered the level of IL-1ß, IL-6, MCP-1, and TNF-α in EG-stimulated kidney tissue and inhibited the protein level of EG-induced TGF-ß1, p-Smad3, and p-Smad2 in a concentration-dependent manner. Conclusion: P. petiolosa extract can improve EG-induced urolithiasis in rats by inhibiting oxidative stress, inflammatory response, and the activation of TGF-ß pathway.

Effect of Rhizoma Drynariae on differential gene expression in ovariectomized rats with osteoporosis based on transcriptome sequencing.

Osteoporosis is increasingly becoming a serious problem affecting the quality of life of the older population. Several experimental studies have shown that Chinese medicine has a definite effect on improving osteoporosis. Based on transcriptome sequencing, we analyzed the differential gene expression and mechanism of the related signaling pathways. Fifteen rats were randomly divided into an experimental group, a model group, and a sham surgery group. The rat model for menopausal osteoporosis was established using an ovariectomy method. One week after modeling, the experimental group was administered(intragastric administration)8.1 g/kg of Rhizoma drynariae, whereas the model and sham groups received 0.9% saline solution twice daily for 12 weeks. Subsequently, the rats were sacrificed, and the left femur of each group was removed for computerized tomography testing, while right femurs were used for hematoxylin and eosin staining. High-throughput RNA sequencing and functional and pathway enrichment analyses were performed. Comparing the gene expression between the experimental and model groups, 149 differential genes were identified, of which 44 were downregulated and 105 were upregulated. The criteria for statistical significance were |log2 Fold Change| > 1 and P < 0.05. Gene ontology analysis showed that the differentially expressed genes were enriched in cell component terms such as cell part and outer cell membrane part, and the genes were associated with cell process, biological regulation, metabolic processes, DNA transcription, and catalytic activity. Enrichment analysis of Kyoto Encyclopedia of Genes and Genomes pathways showed significantly enriched pathways associated with systemic lupus erythematosus, herpes simplex infection, circadian rhythm, vascular smooth muscle contraction, the AGE-RAGE signaling pathway in diabetic complications, and the TNF, Apelin, and Ras signaling pathways. Our results revealed that the Npas2, Dbp, Rt1, Arntl, Grem2, H2bc9, LOC501233, Pla2g2c, Hpgd, Pde6c, and Dner genes, and the circadian rhythm, lipid metabolism, inflammatory signaling pathway, and immune pathways may be the key targets and pathways for traditional Chinese medicine therapy of Rhizoma Drynariae in osteoporosis.

Total flavonoids of Rhizoma drynariae promote angiogenesis and osteogenesis in bone defects.

Bone defects are difficult to heal, which conveys a heavy burden to patients' lives and their economy. The total flavonoids of Rhizoma drynariae (TFRD) can promote the osteogenesis of distraction osteogenesis. However, the dose effect is not clear, the treatment period is short, and the quality of bone formation is poor. In our study, we observed the long-term effects and dose effects of TFRD on bone defects, verified the main ingredients of TFRD in combination with network pharmacology for the first time, explored its potential mechanism, and verified these findings. We found that TFRD management for 12 weeks regulated osteogenesis and angiogenesis in rats with 4-mm tibial bone defects through the PI3K/AKT/HIF-1α/VEGF signaling pathway, especially at high doses (135 mg kg-1  d-1 ). The vascularization effect of TFRD in promoting human umbilical vein endothelial cells was inhibited by PI3K inhibitors. These results provide a reference for the clinical application of TFRD.

Total flavonoids of Rhizoma Drynariae enhances CD31hiEmcnhi vessel formation and subsequent bone regeneration in rat models of distraction osteogenesis by activating PDGF­BB/VEGF/RUNX2/OSX signaling axis.

Total flavonoids of Rhizoma Drynariae (TFRD), extracted from the kidney­tonifying Traditional Chinese medicine Rhizoma Drynariae, can be effective in treating osteoporosis, bone fractures and defects. However, the pharmacological effects of TFRD on the specific vessel subtype CD31hiEmcnhi during distraction osteogenesis (DO) remains unclear. The present study aimed to investigate the effects of TFRD on CD31hiEmcnhi vessels in a rat model of DO. In the present study, tibial DO models were established using 60 rats with a distraction rate of 0.2 mm per day for 20 days. Co­immunofluorescence staining of CD31 and endomucin (Emcn) was conducted to determine CD31hiEmcnhi vessels. Radiographic, angiographic and histological analyses were performed to assess bone and vessel formation. Tube formation, alkaline phosphatase (ALP) and Von Kossa staining assays were performed to test angiogenesis of endothelial precursor cells (EPCs) and osteogenesis of bone marrow­derived mesenchymal stem cells (BMSCs). Additionally, expression levels of platelet­derived growth factor (PDGF)­BB, VEGF, runt­related transcription factor 2 (RUNX2) and Osterix (OSX) were determined by western blotting and reverse transcription­quantitative PCR. The in vivo assays demonstrated that TFRD markedly promoted CD31hiEmcnhi vessel formation during DO, whereas PDGF­BB neutralizing antibody suppressed vessel formation. Furthermore, the ALP, Von Kossa staining and tube formation assays indicated that TFRD notably elevated the angiogenic capacity of EPCs and osteogenic capacity of BMSCs under stress conditions, which was significantly suppressed by blocking PDGF­BB. The protein and mRNA levels of PDGF­BB, VEGF, RUNX2 and OSX were upregulated by TFRD, but downregulated by blocking PDGF­BB. Thus, TFRD could facilitate CD31hiEmcnhi vessel formation and subsequently enhance angiogenic­osteogenic coupling to regenerate bone defects during DO via the PDGF­BB/VEGF/RUNX2/OSX signaling axis, which indicated that CD31hiEmcnhi vessels could be a potential novel therapeutic target for DO, and TFRD may represent a promising drug for promoting bone regeneration in DO by increasing CD31hiEmcnhi vessels.

Total Flavonoids of Drynariae Rhizoma Improve Glucocorticoid-Induced Osteoporosis of Rats: UHPLC-MS-Based Qualitative Analysis, Network Pharmacology Strategy and Pharmacodynamic Validation.

Background: Glucocorticoid-induced osteoporosis (GIOP) is a common form of secondary osteoporosis caused by the protracted or a large dosage of glucocorticoids (GCs). Total flavonoids of Drynariae rhizoma (TFDR) have been widely used in treating postmenopausal osteoporosis (POP). However, their therapeutic effects and potential mechanism against GIOP have not been fully elucidated. Methods: Ultra-high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESIQ-TOF-MS) experiments were performed for qualitative analysis. We performed hematoxylin-eosin (HE) staining and microcomputed tomography (micro-CT) analysis to detect the changes in bone microstructure. The changes in biochemical parameters in the serum samples were determined by performing an enzyme-linked immunosorbent assay (ELISA). The prediction results of network pharmacology were verified via quantitative real-time polymerase chain reaction (qRT-PCR) to elucidate the potential mechanism of TFDR against GIOP. Results: A total of 191 ingredients were identified in vitro and 48 ingredients in vivo. In the in-vivo experiment, the levels of the serum total cholesterol (TC), the serum triglyceride (TG), Leptin (LEP), osteocalcin (OC), osteoprotegerin (OPG), bone morphogenetic protein-2 (BMP-2), propeptide of type I procollagen (PINP), tartrate-resistant acid phosphatase (TRACP) and type-I collagen carboxy-terminal peptide (CTX-1) in the TFDR group significantly changed compared with those in the GIOP group. Moreover, the TFDR group showed an improvement in bone mineral density and bone microstructure. Based on the results of network pharmacology analysis, 67 core targets were selected to construct the network and perform PPI analysis as well as biological enrichment analysis. Five of the targets with high "degree value" had differential gene expression between groups using qRT-PCR. Conclusion: TFDR, which may play a crucial role between adipose metabolism and bone metabolism, may be a novel remedy for the prevention and clinical treatment of GIOP.

Gu Sui Bu (Drynaria fortunei J. Sm.) antagonizes glucocorticoid-induced mineralization reduction in zebrafish larvae by modulating the activity of osteoblasts and osteoclasts.

ETHNOPHARMACOLOGICAL RELEVANCE: Gu Sui Bu (GSB), the dried rhizome of Drynaria fortunei J. Sm., is widely used in traditional Chinese medicine for treating fractures and osteoporosis. Although glucocorticoids are widely prescribed in modern medicine, the efficacy of GSB in treating glucocorticoid-induced osteoporosis (GIOP) remains unclear. AIM OF THE STUDY: GIOP is one of the most prevalent forms of osteoporosis and increases the risk of fracture, which can cause severe complications in elderly people. Safe, efficacious, and cost-effective treatment options for GIOP are thus warranted. The present study investigated the efficacy and mechanism of GSB for treating GIOP. MATERIALS AND METHODS: We established an efficient and robust in vivo GIOP model by optimizing zebrafish larvae rearing conditions and the dose and duration of dexamethasone treatment. Bone calcification was evaluated through calcein staining. To quantify the degree of vertebral mineralization in the larvae, we developed a scoring system based on the rate of vertebral calcification; this system reduced quantification errors among individual zebrafish caused by inconsistencies in staining or imaging parameters. Quantitative real-time polymerase chain reaction was used to access the expression levels of genes essential to the differentiation and function of bone cells. High-performance liquid chromatography was employed to identify naringin in the GSB extract. RESULTS: GSB significantly reversed the dexamethasone-induced calcification delay in zebrafish larvae. GSB enhanced osteoblast activity by increasing the expression of collagen I, osteopontin, and osteonectin and repressed bone resorption by decreasing the expression of matrix metalloproteinases (mmps), including mmp9 and mmp13a. We also identified naringin as one of the constituents of GSB responsible for the herbal extract's anti-GIOP activity. CONCLUSIONS: Using the in vivo zebrafish GIOP model that we established, the efficacy of traditional Chinese medicines in treating GIOP could be systematically investigated. GSB has an osteogenic effect and may thus be an efficacious and cost-effective treatment option for GIOP. Notably, bone resorption activity was found to be retained after GSB treatment, which would be beneficial for maintaining normal bone remodeling.

Study on the Mechanism of Treating Femoral Head Necrosis with Drynariae Rhizoma Based on Network Pharmacology.

Through the network pharmacology thought, the action target of the active ingredients of Drynariae Rhizoma was predicted, and the mapping was combined with the related targets of ONFH, and the key nodes of interaction were identified for enrichment analysis, so as to comprehensively explore the pharmacological mechanism of Drynariae Rhizoma against ONFH. The main active ingredients of Drynariae Rhizoma were screened based on pharmacokinetic characteristics in pharmacokinetic database and analysis platform of TCM system (TCMSP). We used the organic small molecule bioactivity database (PubChem) and Swiss target prediction database to predict related targets based on 2D or 3D structural similarity and then mined the known ONFH therapeutic targets through the Human Mendelian Genetic Database (OMIM) and Pubmed texts. Combined with the predicted targets, String database was imported to construct the OP target interaction network diagram of bone fracture therapy. CytoNCA software was used to topology the key nodes of interaction according to relevant node parameters, and String was imported again to construct the protein interaction network diagram. Finally, biological functions and metabolic pathways of key nodes were analyzed through DAVID database. It was revealed that Drynariae Rhizoma may regulate stem cells, osteoblasts, osteoclasts, and immune cells through multiple pathways, including proliferation, differentiation, immunity, and oxidative stress. Conclusion: Pharmacological studies based on network indicate that Drynariae Rhizoma may participate in the regulation of several major signaling pathways through direct or indirect action targets and affect the proliferation and differentiation of multiple types of cells, thus playing an anti-ONFH role, which provides a scientific basis for explaining the material basis and mechanism of its anti- ONFH.

Network Pharmacology Analysis and Experimental Validation to Investigate the Mechanism of Total Flavonoids of Rhizoma Drynariae in Treating Rheumatoid Arthritis.

Objective: The study aimed to explore the mechanism of total flavonoids of Rhizoma Drynariae (TFRD) in the treatment of rheumatoid arthritis (RA) based on network pharmacology and experimental validation. Methods: The active components of TFRD were identified from TCMSP and TCMID databases. Relevant targets of the active compounds of TFRD and RA-related targets were predicted by public databases online. A component-target (C-T) regulatory network was constructed by Cytoscape. The genes of TFRD regulating RA were imported into STRING database to construct a protein-protein interaction (PPI) network in order to predict the key targets. KEGG enrichment analysis was performed to predict the crucial mechanism of TFRD against RA. The active components of TFRD underwent molecular docking with the key proteins. Collagen-induced arthritis (CIA) model of rats and inflammatory factors-stimulated fibroblast-like synoviocytes were used in vivo and in vitro to validate the efficacy and predicted critical mechanisms of TFRD. Results: Network Pharmacology analysis revealed that TFRD had 14 active compounds, corresponding to 213 targets, and RA related to 2814 genes. There were 137 intersection genes between TFRD and RA. KEGG indicated that therapeutic effects of TFRD on RA involves T cell receptor signaling pathway, Th17 cell differentiation, IL-17 signaling pathway, TNF signaling pathway, MAPK signaling pathway and PI3K/AKT signaling pathway. In vivo experiments suggested TFRD can alleviate the inflammatory response, joint swelling and synovial abnormality of CIA rats. TFRD contributed to the decrease of Th17 cells and the down-regulated secretion of IL-17A and TNF-α of activated lymphocyte in CIA model. In vitro experiments confirmed TFRD can effectively inhibit the inflammatory response of fibroblast-like synoviocytes and suppress the abnormal activation of MAPK, PI3K/AKT and NFκB signaling pathways. Conclusion: The treatment of RA with TFRD is closely related to inhibiting Th17 differentiation and inflammatory response of synoviocytes.

Water Extract of Rhizoma Drynaria Selectively Exerts Estrogenic Activities in Ovariectomized Rats and Estrogen Receptor-Positive Cells.

Our previous study demonstrated that the bone protective actions of herbal medicine Rhizoma Drynariae (Gusuibu, RD) were mainly mediated by flavonoid phytoestrogens via estrogen receptors, raising concerns about the safety of using RD as it may induce estrogen-like risk-benefit profile and interact with other ER ligands, such as selective estrogen receptor modulators (SERMs), when coadministered. The present study evaluated the estrogenic activities of RD and its potential interaction with tamoxifen, a SERM, in estrogen-sensitive tissues by using mature ovariectomized (OVX) rats and ER-positive cells. Similar to but weaker than tamoxifen, RD at its clinical dose dramatically ameliorated OVX-induced changes in bone and dopamine metabolism-related markers in OVX rats. However, tamoxifen, but not RD, induced uterotrophic effects. No significant alteration in mammary gland was observed in OVX rats treated with RD, which was different from the inhibitory actions of tamoxifen. The two-way ANOVA results indicated the interactions between RD and tamoxifen in the bone, brain, and uterus of OVX rats while RD did not alter their responses to tamoxifen. Our results demonstrate that RD selectively exerts estrogenic actions in a different manner from tamoxifen. Moreover, RD interacts with tamoxifen without altering its effects in OVX rats.

Drynaria quercifolia suppresses paracetamol­induced hepatotoxicity in mice by inducing Nrf-2.

Paracetamol is a popular and safe drug preferred by victims of pain or pyrexia; however, its overdose or abuse is a growing concern worldwide. Here the hepatoprotective effect of an ethnomedicinal plant Drynaria quercifolia against paracetamol­induced toxicity in murine model is demonstrated. This fern, native to tropical countries including the Northeast India, is used by local tribes to treat inflammatory conditions. Paracetamol 500 mg/kg body weight was orally administered on alternate days for a period of 21days to mimic a chronic overdose. Drynaria quercifolia acetone extract (DQA) treatment interspaced with paracetamol significantly decreased serum biomarkers of hepatotoxicity (ALT, AST and ALP) renal toxicity (urea, creatinine), lipid peroxidation level, histological damage in liver and kidney. The protein and mRNA expressions of the transcription factor, Nrf2, and its target antioxidant genes (SOD1, CAT and GST) as well as activities of these antioxidant enzymes were downregulated by paracetamol administration but significantly recovered following the DQA treatment (Tab. 3, Fig. 5, Ref. 31). Keywords: acetaminophen/paracetamol, Drynaria quercifolia, renal toxicity, hepatotoxicity, Nrf-2.

Ethanolic extract of Pyrrosia lingua (Thunb.) Farw. ameliorates OVX-induced bone loss and RANKL-induced osteoclastogenesis.

Pyrrosia lingua (Thunb.) Farw is a common plant that has been widely used as a traditional herbal medicine in China and Korea to treat patients suffering from pain, vaginal bleeding and urolithiasis. However, the pharmacological effects of P. lingua on bone remain unknown. We investigated the anti-osteoporotic effects of an ethanolic extract of P. lingua (EEPL). We found that EEPL suppressed osteoclast differentiation by directly acting on osteoclast precursor cells. EEPL suppressed the expression of receptor activator of nuclear factor-κB ligand (RANKL)-induced nuclear factor of activated T cells 1, a major transcription factor for osteoclastogenesis, by inhibiting RANKL-induced expression of aryl hydrocarbon receptor/c-Fos, and activation of nuclear factor-κB and mitogen-activated protein kinases. Moreover, administration of EEPL inhibited trabecular bone loss and weight gain in ovariectomized mice. Furthermore, we identified phytochemicals in EEPL that are known to exert anti-osteoclastogenic or anti-osteoporotic effects using ultra-high-performance liquid chromatography-tandem mass-spectrometry analysis. Overall, the results of this study suggest that EEPL is effective therapeutic candidate that can be used to prevent or treat postmenopausal osteoporosis.

Drynaria fortunei improves lipid profiles of elderly patients with postmenopausal osteoporosis via regulation of Notch1-NLRP3 inflammasome-mediated inflammation.

BACKGROUND: Dyslipidemia is a common comorbidity in elderly patients with postmenopausal osteoporosis (PMOP). Drynaria fortunei (Rhizoma drynariae) is well-known in traditional Chinese medicine for its ability to improve bone mineral density (BMD). However, whether and how Drynaria fortunei improves plasma lipid profiles in elderly PMOP patients remains unclear. METHODS: Eighty elderly female patients with concurrent PMOP and hyperlipemia were randomly assigned to Drynaria fortunei 2(n = 40) or control (n = 40) groups. The clinical efficacies of Drynaria fortunei were evaluated. At 0, 3-, 6-, 9-, and 12-month of follow-up, plasma levels of IL-1ß, IL-18, TNF-α, IL-6, IL-8, and IL-10 were measured using ELISA, whereas PBMC levels of NLRP3, ASC, caspase-1, NF-κB, SIRT1, and Notch1 were measured using RT-qPCR. PBMC isolated from PMOP patients were cultured and treated with Drynaria fortunei to determine its influence on NLRP3 inflammasome and associated cytokines. RESULTS: Drynaria fortunei effectively improved patients' BMD and lipid profiles. IL-1ß, IL-18, TNF-α, IL-6, IL-8 levels, as well as inflammasome-molecules of NLRP3, ASC, caspase-1, and NF-κB increased over time in the control group, but were significantly attenuated with Drynaria fortunei administration. In vitro, Drynaria fortunei suppressed NLRP3 inflammasome and associated cytokines by increasing SIRT1 or decreasing Notch1. Drynaria fortunei had inhibitory effects on NLRP3 inflammasome and Notch1 even when SIRT1 expression was suppressed. CONCLUSIONS: Drynaria fortunei has been demonstrated to significantly improve lipid profiles for elderly PMOP patients. Drynaria fortunei may down-regulate Notch1 independently of SIRT1 to suppress NLRP3 inflammasome-mediated inflammation, thus improving plasma lipid profile.