Enhancing cranial defect repair in rats: investigating the effect of combining Total Flavonoids from Rhizoma Drynariae with calcium phosphate/collagen scaffolds.

OBJECTIVE: To investigate the therapeutic efficacy of total flavonoids of Rhizoma Drynariae (TFRD) in conjunction with a calcium phosphate/collagen scaffold for the repair of cranial defects in rats. METHODS: The subjects, rats, were segregated into four groups: Control, TFRD, Scaffold, and TFRD + Scaffold. Cranial critical bone defects, 5 mm in diameter, were artificially induced through precise drilling. Post-surgery, at intervals of 2, 4, and 8 weeks, micro-CT scans were conducted to evaluate the progress of skull repair. Hematoxylin-eosin and Masson staining techniques were applied to discern morphological disparities, and immunohistochemical staining was utilized to ascertain the expression levels of local osteogenic active factors, such as bone morphogenetic protein 2 (BMP-2) and osteocalcin (OCN). RESULTS: Upon examination at the 8-week mark, cranial defects in the Scaffold and TFRD + Scaffold cohorts manifested significant repair, with the latter group displaying only negligible foramina. Micro-CT examination unveiled relative to its counterparts, and the TFRD + Scaffold groups exhibited marked bone regeneration at the 4- and 8-week intervals. Notably, the TFRD + Scaffold group exhibited substantial bone defect repair compared to the TFRD and Scaffold groups throughout the entire observation period, while histomorphological assessment demonstrated a significantly higher collagen fiber content than the other groups after 2 weeks. Immunohistochemical analysis further substantiated that the TFRD + Scaffold had augmented expression of BMP-2 at 2, 4 weeks and OCN at 2 weeks relative to other groups. CONCLUSIONS: The synergistic application of TFRD and calcium phosphate/collagen scaffold has been shown to enhance bone mineralization, bone plasticity, and bone histomorphology especially during initial osteogenesis phases.

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.

Total Flavonoids from Rhizoma Drynariae (Gusuibu) Alleviates Diabetic Osteoporosis by Activating BMP2/Smad Signaling Pathway.

INTRODUCTION: Diabetic osteoporosis (DOP) is a widespread public health problem. The flavonoids of Rhizoma Drynariae (RDF) have a clear preventive and therapeutic effect on osteoporosis (OP), but it is not yet clear whether RDF has an anti-DOP and whether its mechanism is related to the activation of the BMP2/Smad signaling pathway. The current study aimed to study this effect of RDF in DOP rats and the possible involvement of the BMP2/Smad signaling pathway activation. METHODS: Following intragastric administration of RDF for 12 weeks, the body weight, blood glucose, and the bone histopathological changes detected by hematoxylin-eosin (H&E) and calcein staining were monitored, while bone parameters were regularly assessed from observations made by micro-CT. At the end of the experiment, the expression of Bmp2, Bmpr1a, Runx2, and Smad4/5 genes was detected by real-time PCR (RT-PCR). Meanwhile, western blotting or immunohistochemical staining monitored the protein expressions of BMP2, RUNX2, and SMAD5 in the bone. RESULTS: The results firstly indicated that RDF significantly alleviated the signs and symptoms of DOP, which manifested as improved body weight and blood glucose. As obtained from the results of histopathology and micro-CT, RDF could promote the formation of bone trabeculae and alter several the bone microstructure parameters, including an increase in the bone volume/total volume (BV/TV), connective density (Conn-Dens), and trabecular bone number (Tb.N), as well as a decrease in the trabecular spacing (Tb.Sp). The western blotting analysis and RT-PCR results also confirmed that RDF could markedly increase the mRNA expression levels of Bmp2, Bmpr1α, Smad4, Runx2, and Smad5 in the bone, as well as the corresponding protein expression levels of BMP2, RUNX2, and SMAD5. These results reveal that RDF can activate the BMP2/Smad signaling pathway, thus promoting bone remodeling in DOP rats. CONCLUSION: RDF can increase bone trabeculae and bone mineral density by promoting bone formation and inhibiting bone absorption, thereby playing a role in improving DOP. This effect is related to the regulation of the BMP2/Smad signaling pathway.

Drug Value of Drynariae Rhizoma Root-Derived Extracellular Vesicles for Neurodegenerative Diseases Based on Proteomics and Bioinformatics.

Extracellular vesicles (EVs) are nano-sized membrane vesicles released by various cell types. Mammalian EVs have been studied in-depth, but the role of plant EVs has rarely been explored. For the first time, EVs from Drynariae Rhizoma roots were isolated and identified using transmission electron microscopy and a flow nano analyzer. Proteomics and bioinformatics were applied to determine the protein composition and complete the functional analysis of the EVs. Seventy-seven proteins were identified from Drynariae Rhizoma root-derived EVs, with enzymes accounting for 47% of the proteins. All of the enzymes were involved in important biological processes in plants. Most of them, including NAD(P)H-quinone oxidoreductase, were enriched in the oxidative phosphorylation pathway in plants and humans, and Alzheimer's disease, Huntington's disease, and Parkinson's disease, which are associated with oxidative stress in humans. These findings suggested that EVs from Drynariae Rhizoma roots could alleviate such neurological diseases and that enzymes, especially NAD(P)H-quinone oxidoreductase, might play an important role in the process.

Effect of flavonoids from Rhizoma Drynariae on osteoporosis rats and osteocytes.

In this experimental study, we evaluated the protective effects and the safety of main flavanones derived from Rhizoma Drynariae (Gusuibu) in vitro and in vivo. The MTT assay showed that compared with vehicle treatment, treatment with such flavanones as neoeriocitrin, naringin, and naringenin significantly promoted the viability of osteocyte-like cells. Quantitative real-time PCR showed that neoeriocitrin and naringin significantly attenuated mRNA expressions of RANKL and SOST in osteocyte-like cells. In rats with retinoic acid-induced osteoporosis, total flavonoid of Rhizoma Drynariae (TFRD), naringin, and naringenin significantly increased the number of trabeculae and improved trabecular bone structure compared with no treatment, without affecting liver and renal function. In addition, naringenin and naringin administration significantly increased bone mineral density of femur neck and femur shaft compared with the osteoporotic model rats. Western blot analysis showed that naringenin and naringin significantly attenuated protein expressions of bone resorption-related factors (TRAP, RANKL and RANK), and inhibited sclerostin expression compared with the osteoporotic model rats. On the other hand, naringin markedly increased protein expressions of ALP and PTH1R, and TFRD and naringenin also promoted PTH1R expression compared with the model rats. In conclusion, such flavanones as naringenin and naringin exhibited antiresorptive properties, and naringin particularly showed potential benefits for osteoporosis treatment.

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.

Liglaurates A-E, cytotoxic bis(lauric acid-12yl)lignanoates from the rhizomes of Drynaria roosii Nakaike.

Five undescribed bis(lauric acid-12-yl)lignanoates, liglaurates A-E, along with the known methyl and glyceryl 12-caffeoyloxylaurates were isolated from the rhizomes of Drynaria roosii Nakaike. Their structures including absolute configurations were determined by HRESIMS, NMR techniques, and ECD calculation. Liglaurates A-D were isolated as the racemates, among which (±)-liglaurate A and (±)-liglaurate B were synthesized by a metal-mediated oxidative coupling reaction and further resolved as the enantiomerically pure compounds. Liglaurates (+)-A, (-)-A, (+)-B, (-)-B, (±)-C and (±)-D exhibited remarkable cytotoxic activities against HeLa cell line, with the IC50 values of 0.11 ± 0.02, 0.24 ± 0.01, 0.02 ± 0.00, 0.13 ± 0.02, 0.34 ± 0.07 and 0.17 ± 0.01 µM, respectively.

Receptor-mediated biological effects of extracts obtained from three Asplenium species.

This study reports the effects of aqueous extracts obtained from three fern species of Bulgarian origin: Asplenium ceterach L., Asplenium scolopendrium L., and Asplenium trichomanes L. on the contractility and bioelectrogenesis of rat gastric smooth muscle tissues. In the concentration range 0.015-0.150 mg/mL the three extracts contracted smooth muscle tissues in a concentration-dependent manner. The contractions caused by A. ceterach L. and A. scolopendrium L. extracts (0.150 mg/mL) were reduced by ketanserin (5 × 10-7 and 5 × 10-6 mol/L), an antagonist of serotonin 5-HT2 receptor. The contraction evoked by A. trichomanes L. (0.150 mg/mL) was significantly reduced by 1 × 10-6 mol/L atropine, an antagonist of muscarinic receptors, and turned into relaxation against the background of 3 × 10-7 mol/L galantamine. After combined pretreatment with galantamine and l-arginine (5 × 10-4 mol/L), this relaxation become more pronounced. The study demonstrates that constituents of A. ceterach L. and A. scolopendrium L. extracts act as agonists of 5-HT2 receptors and cause contraction by activating serotonergic signaling system. A. trichomanes L.-induced reaction is an additive result of two opposite-in-character effects. The dominant contraction is initiated by inhibition of acetylcholinesterase activity. The relaxation develops with pre-inhibited acetylcholinesterase, it is significantly potentiated by l-arginine, and therefore associated with nitrergic signaling pathway.

Total flavonoids of Rhizoma drynariae ameliorate steroid­induced avascular necrosis of the femoral head via the PI3K/AKT pathway.

Steroid­induced avascular necrosis of the femoral head (SANFH) is a common orthopaedic disease that is difficult to treat. The present study investigated the effects of total flavonoids of Rhizoma drynariae (TFRD) on SANFH and explored its underlying mechanisms. The SANFH rat model was induced by intramuscular injection of lipopolysaccharides and methylprednisolone. Osteoblasts were isolated from the calvariae of neonatal rats and then cultured with dexamethasone (Dex). TFRD was used in vitro and in vivo, respectively. Haematoxylin and eosin staining was used to assess the pathological changes in the femoral head. Terminal deoxynucleotidyl transferase­mediated deoxyuridine triphosphate nick end labelling assay and flow cytometry were conducted to detect apoptosis of osteoblasts. The 2',7'­dichlorofluorescein­diacetate staining method was used to detect reactive oxygen species (ROS) levels in osteoblasts and the 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide assay was used to detect osteoblast proliferation. The expression of caspase­3, Bax, Bcl­2, VEGF, runt­related transcription factor 2 (RUNX2), osteoprotegerin (OPG), osteocalcin (OCN), receptor activator of nuclear factor κB ligand (RANKL) and phosphoinositide 3­kinase (PI3K)/AKT pathway related­proteins were detected via western blotting. It was found that TFRD reduced the pathological changes, inhibited apoptosis, increased the expression of VEGF, RUNX2, OPG and OCN, decreased RANKL expression and activated the PI3K/AKT pathway in SANFH rats. TFRD promoted proliferation, inhibited apoptosis and reduced ROS levels by activating the PI3K/AKT pathway in osteoblasts. In conclusion, TFRD protected against SANFH in a rat model. In addition, TFRD protected osteoblasts from Dex­induced damage through the PI3K/AKT pathway. The findings of the present study may contribute to find an effective treatment for the management of SANFH.

Evaluation of Cytotoxic Effect of Silver Nanoparticles (AgNP) Synthesized from Phlebodium aureum (L.) J. Smith Extracts.

BACKGROUND: Chemical synthesis methods are adverse in the medicinal field as they produce toxins on the surface area whereas green synthesis provide advancement are cost effective, environment friendly, can be easily scaled up for large scale synthesis. Silver and silver nanoparticles have an important application in the medical industry, such as tropical ointments which are used to prevent infection against burn and open wounds. There is no report on the green synthesis from Phlebodium aureum (L.) J. Smith. OBJECTIVE: The present study was aimed to synthesize silver nano-particles using Phlebodium aureum (L.) J. Smith extracts by green approach and to screen their cytotoxicity. METHODS: The synthesized AgNPs of P. aureum were characterized by FT-IR, SEM and XRD. The cytotoxicity of the aqueous extracts and AgNPs of P. aureum was determined. RESULTS: The silver nanoparticle synthesis was confirmed by color change from yellow to dark brown and absorption peak at 460 nm. FT-IR analysis confirmed the capping by proteins and other metabolites. XRD analysis confirmed the existence of silver nanaoparticles with a peak at 46.253°. The dose dependent cytotoxicity was observed in the aqueous and silver nanoparticles of P.aureum. CONCLUSION: The present study gave a simple and cheap route to synthesize the AgNPs using aqueous extracts of P. aureum. The studied extracts of P. aureum can be considered as a promising candidate for a plant-derived anti-tumour compound.

Effects of Drynaria Total Flavonoid on the Microstructure of the Mandible in Ovariectomized Rats.

BACKGROUND The aim of this study is to investigate the effects of Drynaria total flavonoids (DTF) on mandible microarchitecture, serum estrogen (E2), osteoprotegerin (OPG), and receptor activator of nuclear factor kappa-B ligand (RANKL) levels in an ovariectomy-induced osteoporosis rat model. MATERIAL AND METHODS Thirty female Sprague-Dawley rats were divided into 5 groups (n=6 per group): sham surgery, ovariectomy (OVX), and low-dose, middle-dose, and high-dose DTF. Mandibular osteoporosis was induced by ovariectomy; an equal amount of ovary-sized fat tissue was removed from the sham group. The DTF-treated groups were given DTF gavage at different doses for 12 weeks; the sham and OVX groups were given saline. After the treatment phase, the effects of DTF on the microarchitecture of the mandible were evaluated by measuring bone density, maximum load, morphometric parameters, and histopathological alterations. Serum E2, OPG, and RANKL levels were measured. RESULTS The OVX group showed obvious osteoporosis in the mandible and decreased serum E2 levels and OPG/RANKL ratio. The low-dose group did not show significant improvement in mandibular microstructure. The middle-dose group showed significantly ameliorated osteoporosis. The high-dose group had further improvement in bone microstructures and increase of OPG/RANKL over the middle-dose group. Furthermore, ovariectomy significantly decreased serum E2, but DTF treatment failed to restore serum E2 levels. CONCLUSIONS Ovariectomy can cause significant bone loss in the rat mandible and a decrease in serum E2 and OPG/RANKL. DTF significantly improved the mandibular microstructure and restored OPG/RANKL balance, but it did not restore the decreased serum E2 concentration following ovariectomy.

Effect of total flavonoids of Rhizoma Drynariae in thiram induced cytotoxicity of chondrocyte via BMP-2/Runx2 and IHH/PTHrP expressions.

Tibial Dyschondroplasia (TD) is a prevailing skeletal disorder that mainly affects rapidly growing avian species. It results in reduced bone strength, lameness and an increase risk of fragility fractures. Total flavonoids of Rhizoma drynariae (TFRD) have been used as an effective treatment of different bone diseases in humans. The current in vitro study was conducted to explore the therapeutic effect of TFRD on thiram-induced cytotoxicity in avian growth plate cells via bone morphogenetic protein-2/runt related transcription factor-2 (BMP-2/Runx2) and Indian hedgehog/Parathyroid hormone-related peptide (IHH/PTHrP) expressions. Chondrocytes were isolated, cultured and refined from chicken's tibial growth plates in a special medium. Then chondrocytes were treated with sublethal thiram having less concentration (2.5 µg/mL) to induce cytotoxicity of chondrocyte, and then treated with providential doses (100 µg/mL) of TFRD. Thiram caused distorted morphology of chondrocytes, nuclei appeared disintegration or lysed along with decreased expressions of BMP-2/Runx2 and IHH/PTHrP. TFRD administration not only enhanced the viability of chondrocytes by itself, but also well restored the damage caused by thiram on growth plate chondrocytes by significantly up-regulating the expressions of BMP-2/Runx2 and IHH/PTHrP. Therefore, this study provides a novel insight into the further treatment of TD and other skeletal ailments and lays the foundation for prevention and treatment.

Dietary supplementation of total flavonoids from Rhizoma Drynariae improves bone health in older caged laying hens.

Caged layer osteoporosis (CLO) is a common bone metabolism diseases and poses a great threat to the production of laying hens. So far, there is no effective nutrition intervention to prevent CLO. The objective of this study was to evaluate the effects of dietary total flavonoids from Rhizoma Drynariae (TFRD), a Chinese herbal, on bone health, egg quality, and serum antioxidant capacity of caged laying hens. A total of two hundred sixteen, 54-wk-old Lohmann Pink-shell laying hens at were allocated to 3 groups with 6 replicates of 12 hens per replicate. The control group was fed a basal diet (BD) and 2 treatment groups additionally supplied with 0.5 or 2.0 g/kg TFRD, respectively. Results showed that supplying 2.0 g/kg TFRD enhanced the activities of serum total antioxidant capacity (P < 0.01) and glutathione peroxidase (P < 0.05) and had higher femur and tibia bone mineral density (both P < 0.05) compared with the control group. Dietary 2.0 g/kg TFRD also reduced the activities of serum alkaline phosphatase (P < 0.01), tartrate resistant acid phosphatase (P < 0.01), and the contents of osteocalcin (P < 0.01). Furthermore, tibia histomorphology observation showed that the microstructure of bone tissue was improved after TFRD treatment. Egg quality was not affected by TFRD while the egg weight significantly increased (P < 0.01). These findings suggested that TFRD has beneficial effects on bone health in older caged laying hens.

Preparation and antioxidant study of silver nanoparticles of Microsorum pteropus methanol extract.

This study reports a preparation of silver nanoparticles (SNPs) using Microsorum pteropus methanol extract, as a new approach in the development of therapeutic strategies against diseases caused by oxidative stress, reactive oxygen, and nitrogen species. During the effort of extraction and isolation from M. pteropus, X-ray single-crystal structural analysis of sucrose was succeeded. 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and hydrogen peroxide scavenging assay were used to confirm the antioxidant potential. Preparation of SNPs was confirmed by ultraviolet-visible (UV-Vis) spectra with peaks between 431 and 436 nm. Infrared (IR) analysis showed OH, NH functional groups of alcohol, phenol, amine, and aliphatic CH stretching vibrations of hydrocarbon chains of the synthesized nanoparticles. The antioxidant properties of the SNPs significantly showed DPPH reduction with an IC50 value of 47.0 µg/mL and hydrogen peroxide scavenging activity with an IC50 value of 35.8 µg/mL, and hence, indicating their capability to eliminate potentially damaging oxidants involved in oxidative stress and their related diseases.

Chemical Components and Antibacterial Activity of the Essential Oil of Six Pyrrosia Species.

The present study was aimed at analyzing the chemical components of the essential oil from six Pyrrosia species by GC/MS and evaluating their in vitro antibacterial activities. Seventy volatile compounds were identified in the essential oil of six Pyrrosia samples. The identified volatile components were divided into following nine categories: aldehydes, terpenoids, fatty acids, ketones, furans, hydrocarbons, alcohols, esters, and phenols. The major components of the essential oil were 2,4-pentadienal, phytol and nonanal. The antimicrobial assays showed that the essential oils from Pyrrosia samples exhibited a broad-spectrum antimicrobial activity. However, P. lingua had the highest antibacterial activity against Staphylococcus aureus (ATCC 25923) with a minimum inhibitory concentration (MIC) of 2.5 µL/mL. This article is the first report of the chemical components and antimicrobial activity of the essential oil from six Pyrrosia species, which will lay the foundation for developing medicinal resources from Pyrrosia fronds.

Naringin induces endoplasmic reticulum stress-mediated apoptosis, inhibits ß-catenin pathway and arrests cell cycle in cervical cancer cells.

Naringin is a promising anticancer bioflavonoid phytochemical, mainly extracted from citrus fruits. This study evaluates the antiproliferative effect and the cell death mechanism induced by naringin on cervical cancer (CC) cells. Our results demonstrated that naringin exerts significant inhibition in cell viability and exhibits IC50 value 745, 764, 793 µM against C33A, SiHa, and HeLa cells respectively. Annexin V FITC and immunoblotting analysis reveal significant apoptosis induction in cells exposed to higher doses naringin. Mechanistically, naringin induces endoplasmic reticulum (ER) stress-associated cell killing in CC cells. Naringin increases the protein expression of ER stress sensors, phosphorylates eIF2α by and activates apoptosis-associated protein CHOP and other associated proapoptotic proteins (PARP1 and caspase-3). Intriguingly, pre-treatment with of ER stress inhibitor (salubrinal), reverses the apoptotic effect exerted by naringin. Additionally, the naringin abrogates the ß-catenin pathway by decreasing the protein expression as well as phosphorylation of ß-catenin (Ser576) and GSK-3ß (Ser9) and simultaneously triggers cell cycle arrest at a G0/G1 phase by increasing the expression of cell cycle checkpoint proteins p21/cip and p27/kip. Naringin induces ER stress-mediated apoptosis and simultaneously abrogates Wnt/ß-catenin signaling which eventually triggers the arrest of the cell cycle at a G0/G1 phase in CC cells.

A study of Drynaria fortunei in modulation of BMP­2 signalling by bone tissue engineering

Background/aim: Drynaria fortunei (Gusuibu; GSB) is a popular traditional Chinese medicine used for bone repair. An increasing number of studies have reported that GSB induces osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). These results provide insight into the application of GSB for bone tissue engineering techniques used to repair large bone defects. However, few studies have described the molecular mechanisms of GSB. Materials and methods: In the present study, the effects of GSB and naringin, a marker compound, on the binding of BMP-2 to BMPR and BMP-2-derived signal transduction were investigated using surface plasmon resonance (SPR) and coculturing with BMPR- expressed cell line, C2C12, respectively. Furthermore, naringin was also used to prepare naringin contained scaffolds for bone tissue engineering. The physical and chemical properties of these scaffolds were analysed using scanning electron microscopy (SEM) and highperformance liquid chromatography (HPLC). These scaffolds were cocultured with rabbit BMSCs in vitro and implanted into rabbit calvarial defects for bone repair assessment. Results: The results showed that GSB and naringin affect the binding of BMP and BMPR in SPR experiments. GSB is a subtle BMP modulator that simultaneously inhibits the binding of BMP-2 to BMPR-1A and enhances its binding to BMPR-1B. In contrast, naringin inhibited BMP-2 binding to BMPR-1A. In vitro studies involving the phosphorylation of signals downstream of BMPR and Smad showed that GSB and naringin affected stem cell differentiation by inhibiting BMPR-1A signalling. When using GSB for bone tissue engineering, naringin exhibited a higher capacity for slow and gradual release from the scaffold, which promotes bone formation via osteoinduction. Moreover, control and naringin scaffolds were implanted into rabbit calvarial defects for 4 weeks, and naringin enhanced bone regeneration in vivo significantly. Conclusions: GSB and its marker compound (naringin) could inhibit the binding of BMP-2 and BMPR-1A to control cell differentiation by blocked BMPR-1A signalling and enhanced BMPR-1B signalling. GSB and naringin could be good natural BMP regulators for bone tissue engineering.