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Objective:To study the effect of total flavonoids of Rhizoma Drynariae on learning and memory impairment mice induced by sodium nitrite. Methods:75 mice were divided into blank group, model group, Kangnaoshuai capsule group, Rhizoma Drynariae total flavonoids group and Rhizoma Drynariae total flavonoids+inhibitor group according to the random number table method, with 15 mice in each group. The Kangnaoshui Capsule group was administered with Kangnaoshui Capsule 585 mg/kg, the Rhizoma Drynariae total flavonoids group was administered with the Rhizoma Drynariae total flavonoids 97.5 mg/kg, the Rhizoma Drynariae total flavonoids group and the inhibitor group were administered with the Rhizoma Drynariae total flavonoids by intragastric administration 97.5 mg/kg, and intraperitoneal injection of 0.072 mg/kg ICI182780 for 21 days, once a day. The model was established on the 22nd day. Except for the blank group, the other mice were injected with sodium nitrite intraperitoneally to replicate the mice model with impaired learning and memory capability. The learning and memory capabilit of mice were detected with water maze method, and the estrogen receptor in hippocampus was detected by immunohistochemistry β (estrogen receptor β, ERβ). The expression of ERβ in hippocampus and the expression of phosphorylated P38 (P-P38) and the protein contents of B-cell lymphoma-2 (Bcl-2), Bcl-2 associated death promoter (Bad) and Caspase-3 in the apoptotic system was detected by Western blot. The kit was used to detect MDA,SOD and NO protein content in hippocampus. Results:The latency of Rhizoma Drynariae total flavonoids group was significantly shorter than the model group, the number of crossing platform and the residence time in the target quadrant were significantly increased ( P<0.01); The expression of ERβ Protein in mice hippocampus (0.371 ± 0.010 vs. 0.124 ± 0.009), Bcl-2 protein (1.146 ± 0.028 vs. 0.726 ± 0.016) and the contents of SOD [(153.657 ± 6.385) U/mg vs. (67.719±5.845) U/mg] increased significantly ( P<0.01); The expression of P-P38/P38 protein (0.412 ± 0.043 vs.0.806 ± 0.069), Bad protein (0.421 ± 0.010 vs.0.633 ± 0.010), Caspase-3 protein (0.923 ± 0.042 vs.1.437 ± 0.033), and the content of MDA [(8.669 ± 0.662) nmol/mg vs. (11.772 ± 1.054) nmol/mg] and NO [(4.259 ± 0.225) nmol/mg vs. (10.805 ± 0.415) nmol/mg] decreased significantly ( P<0.01). In addition, ER blocker can antagonize the above recovery and improvement effects of Rhizoma Drynariae total flavonoids group. Conclusion:Rhizoma Drynariae total flavonoids can regulate memory impairment, inhibit neuronal apoptosis and reduce oxidative stress in sodium nitrite model mice through ER-P38/MAPK signal pathway.
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BACKGROUND: Total flavonoids of Rhizoma Drynariae can be used to prevent and treat osteoporosis, but its mechanism of action is mostly focused on osteoblasts rather than osteoclast differentiation. OBJECTIVE: To investigate the effect of Rhizoma Drynariae on osteoclast differentiation through T-cell nuclear factor 1 (NFATc1) signaling pathway. METHODS: Forty Sprague-Dawley rats were randomly divided into four groups. Total flavonoids of Rhizoma Drynariae were intragastrically administered at 0, 11.6, 34.8 and 104.4 g/kg, respectively. Control serum and low-, middle-, high-concentration drug-containing sera were obtained. Bone marrow macrophages were isolated from 5-week-old rats and the effects of different drug-containing sera on macrophage activity were detected by cell counting kit-8 method. The macrophages were divided into low-, middle-and high-concentration groups, negative control group and blank group, with 5 multiple holes in each well. The low, middle and high concentration of drug-containing serum, negative control serum culture medium and normal culture medium were added respectively. All culture media were used to induce osteoclast differentiation with 20 μg/L macrophage colony-stimulating factor and 100 μg/L receptor activator of nuclear factor kappa B ligand. The number of osteoclasts and fusion index were detected by tartrate-resistant acid phosphatase staining 7 days after induction of differentiation. The expressions of c-Fos, Fra-1, Fra-2, NFATc1 and cathepsin K were detected by real-time fluorescence quantitative PCR and western blot at 7 days after differentiation. The number of bone resorption lacunae and the proportion of lacunae area in osteoclasts after 14 days of differentiation were detected by bone fragment absorption lacunae test. The study protocol was approved by the Animal Experiment Ethics Committee of Anyang District Hospital of Puyang City with approval No. PYSAYDQ-2019096. RESULTS AND CONCLUSION: Cell counting kit-8 results showed that there was no significant change in macrophage activity after intervention with different concentration of sera containing Rhizoma Drynariae. The morphology of macrophages was regular before differentiation. After differentiation, osteoclasts were identified by tartrate-resistant acid phosphatase staining. Compared with the blank group and negative control group, the number of osteoclasts, fusion index, number of bone resorption lacunae and proportion of lacunae area and the relative expressions of c-Fos, Fra-1, Fra-2, NFATc1, cathepsin K mRNA and protein in the low-, middle-and high-concentration groups decreased significantly (P middle-concentration group > high-concentration group, and there were significant differences between different concentration groups (P < 0.05). In conclusion, Rhizoma Drynariae may inhibit the differentiation of rat macrophages into osteoclasts through NFATc1 signaling pathway, and the degree of differentiation inhibition is related to the serum concentration of Rhizoma Drynariae.
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Objective:To use polylactic-co-glycolic acid(PLGA) as vector material to prepare the compound total flavonoids of Rhizoma Drynariae(TFRD) and total flavonoids of Chrysanthemum(TFC) sustained release microcapsule TF-PLGA microcapsules,and to investigate the the best preparation technique of TF-PLGA microcapsules and their sustained release characteristics in vitro.Methods:The TF-PLGA microcapsules were prepared with TFRD,TFC,and PLGA by emulsifying-solvent evaporation technique under certain conditions.With the encapsulation efficiency(EE) as the evaluation indicator,the optimal formulation was verified by single factor experiment and orthogonal design;the general morphology,the particle size and distribution of the microcapsules were observed by light microscope(LM) and scanning electron microscope(SEM);the cumulative drug release rate of TF-PLGA microcapsules was detected by constant temperature commotion method in vitro and the release curves of the TF-PLGA were drawn.Results:The optimal prescription was as follows:the concentration of PLGA was 140 g·L-1,oil phase volume was 1.4 mL,emulsifying speed was 900 r·min-1,emulsifying time was 5 min,the average EE was(83.89±2.30)%,and the average drug loading rate(DL) was(5.90±0.07)%.The LM and SEM resluts showed that the TF-PLGA microcapsules presented as round ball,the average particle size was(44.34±14.68)μm,and the distribution was relatively narrow.The drug release in vitro results showed that the initial drug release rate(24 h)was about 40%,and the cumulative drug release rate was over 90% after 50 d.Conclusion:The TF-PLGA sustained release microcapslue has better drug-loaded and sustained-release effects with simple preparation technique and better repeatability.
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Objective To explore the effects of flavonoids of Rhizoma Drynariae on the formation of blood vessels in the induced membrane by Masquelet technique. Methods Seventy-two SD rats were randomly divided into 4 groups,namely model group,and high-,middle-and low-dose drug groups,18 rats in each group. Rat model of critical- sized femoral defect was built,and then polymethyl methacrylate (PMMA)bone cement spacer was inserted into the bone defect to induce the formation of membrane. From the first day after surgery , the rats in high-,middle-and low-dose drug groups were given gastric gavage of 0.44,0.22,0.11 g·kg-1·d-1 of Rhizoma Drynariae flavonoids, respectively, and the rats in the model group were given the same volume of normal saline. After 6-week medication,the pathologic features of bone cement- induced membrane were observed by Haematoxilin-Eosin(HE)staining,the contents of transforming growth factor(TGF)-β1 and vascular endothelial growth factor(VEGF)proteins in the induced membrane were tested by enzyme-linked immunosorbent assay (ELISA),and the mRNA levels of TGF-β1 and VEGF in the induced membrane were determined by real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Results More blood vessels in the induced membrane of the high-dose group were found than those of the other groups under the light microscope. The protein and mRNA expression levels of TGF-β1 and VEGF in the induced membrane of the 3 drug groups were much higher than those of the model group(P < 0.05). Except for the VEGF mRNA expression level, the changes of other indexes were dose-dependent. Conclusion Flavonoids of Rhizoma Drynariae are effective on enhancing the protein and mRNA expression levels of TGF-β1 and VEGF in the induced membrane, and can accelerate the vascularization,which promotes the reconstruction of bone defect.
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Objective To establish a distraction osteogenesis SD rat model for exploring the effect of total flavonoids from Rhizoma Drynariae on the quality of rat bone formation in the process of distraction osteogenesis from the perspective of tomography.Methods Twenty-four rats were randomly divided into experimental group and control group after successful modeling,and were respectively given intragastric administration with total flavonoids from Rhizoma Drynariae(in dose of 77.125 mg/kg) and saline for 8 weeks.Seven days after operation,the tibias of all the rats were given distraction at the speed of 0.2 mm for 20 days.Eight weeks after the operation,X-ray examination for all the rats was performed,and then the rats were killed for obtaining the tibia samples for Micro-CT scanning.The differences of X-ray Lane-Sandhu scores,the bone mineral density and bone mineral content were compared between the two groups.Results The X-ray Lane-Sandhu scores,the bone mineral density and bone mineral contents showed by Micro-CT scanning in the experimental group were higher than those in the control group,and the differences were statistically significant (P ＜ 0.05).Conclusion The total flavonoids from Rhizoma Drynariae can improve the quality of bone formation in the process of distraction osteogenesis in rats.
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Aim To investigate the drug-containing serum of Rhizoma drynariae on osteogenesis differentiation of bone marrow mesenchymal stem, and discuss the possible mechanism.Methods BMSCs were cultured in media with different concentrations of medicine containing serum.BMSCs proliferation ability was detected in 3,5,7,9 days by CCK-8.ALP activity was detected after 7,10,14 days′ induction.After 3 weeks culturing, alizarin red staining was performed to observe the formation of calcium nodules.The expression of β-catenin,LRP5,RUNX-2 and Osteriex mRNA were detected using RT-PCR.The protein expression of β-catenin,LRP5 was detected using Elisa method.Results Rhizoma drynariae drug-containing serum could obviously promote the proliferation of BMSCs and calci-fied nodule formation.Besides, the ALP activity was improved in a certain period of time.The expression of β-catenin,LRP5,GSK-3β,RUNX-2 and Osteriex mRNA were significantly up-regulated,and the protein expression of β-catenin,LRP5 was up-regulated too.The expression of GSK-3β was down-trgulated.Conclusions Rhizoma drynariae drug-containing serum promotes mineralization and osteogenic differentiationof BMSCs, and the mechanism is closely related with activating WNT/beta-catenin signaling pathway, raising the beta-catenin, LRP5, RUNX-2, and Osteriex mRNA expression, beta-catenin, LRP5 protein expression,and down-regulation of GSK-3β mRNA expression.
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Objective To study the effects about rhizoma drynariae poly (lactic-co-glycolic acid) (DR-PLGA) /Calcium phosphate cement (CPC) composite scaffold on treating rabbit femoral bone defect.Methods Eight Newzealand rabbits were randomly divided into experiment group(n =4) and control group (n =4).The preparation of rabbit femoral bone defect model,separately implanted DR-PLGA/CPC scaffold and PLGA/CPC scaffold.After 4,8 weeks,we took out the materials,observed with X-ray,gross anatomy,histology observation to evaluate the osteogenetic activity,the effect of accelerating the healing of bone defect.Resuits At the 4th week and 8th week after implantation,the effect of promoting fracture healing and osteogenic activity of the experiment group were greater than those in the control group.Conclusions DR-PLGA combined with CPC could induce new bone formation,promote the healing of rabbit femoral defect.
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Objective To investigate the effect of total flavones of Rhizoma Drynariae on femur distraction osteogenesis in the rabbits. Methods Thirty-two healthy rabbits were randomly divided into treatment group and control group, 16 rabbits in each group. The femoral fracture was treated with unilateral femoral distraction and was fixed with a self-made distraction instrument. After 7-day intermittent period, the fractured femur was distracted at a rate of 1 mm/d, twice a day for 10 continuous days. The treatment group was fed with total flavones of Rhizoma Drynariae from the first post-operative day to the end of the experiment. And then all of the animals were sacrificed after fixation for 28 days. The bone specimens were used for histological observation and immunohistochemical detection. Results The area of mature bone in the newborn bone tissue of the treatment group was increased, and osteoblasts number and the percentage of trabecular bone area were significantly higher than those of the control group . The bone morphogenetic protein-2 (BMP-2) and transforming growth factor-β1 (TGF-β1) were stained brown deeply, the staining degree being stronger than that of the control group. Conclusion Rhizoma Drynariae total flavones can effectively accelerate the formation and maturation of newborn bone tissue during bone distraction.
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Objective To investigate fingerprint analysis and quantification of Rhizoma Drynaria. Methods HPLC fingerprints were carried out at 30° on a Zirchrom C18 column (4.6 mm×200 mm, 5 µm). The mobile phase was methanol (A) and phosphoric acid aqueous solution (pH3.6, B). Gradient programmer was performed in linear gradient The chromatogram was monitored at a wavelength of 283 nm throughout the experiment and the chromatographic peaks were obtained, ranging from 200 nm to 400 nm. The injection volume was 10 µl. Then quantitative analyses were carried out at 30$ on a Kromasil C18 column (250 mm×4.6 mm,5 µm). Results In fingerprint analysis, plant materials from 16 regions were analyzed under the optimized HPLC conditions and 12 peaks were selected as characteristic peaks Compared with the reference standards, 6 major chromatographic peaks were characterized and identified, with sum of peaks area over 70% according to area normalization method. Additionally, the similarity analysis and HCA analysis were performed and the results got mutual authentication In quantitative analysis, the four compounds showed good regression (r>0.9995) within test ranges and the recovery of the method was in the range of 97.9%-103.7%. Conclusion The results revealed that the method of reference chromatographic fingerprints combined with multiple compounds determination could be used as an efficient strategy for systematic quality evaluation of Rhizoma Drynariae.
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Objective To investigate fingerprint analysis and quantification of Rhizoma Drynaria. Methods HPLC fingerprints were carried out at 30℃on a Zirchrom C18 column (4.6 mm×200 mm, 5μm). The mobile phase was methanol (A) and phosphoric acid aqueous solution (pH3.6, B). Gradient programmer was performed in linear gradient. The chromatogram was monitored at a wavelength of 283 nm throughout the experiment and the chromatographic peaks were obtained, ranging from 200 nm to 400 nm. The injection volume was 10μl. Then quantitative analyses were carried out at 30℃on a Kromasil C18 column (250 mm×4.6 mm,5μm). Results In fingerprint analysis, plant materials from 16 regions were analyzed under the optimized HPLC conditions and 12 peaks were selected as characteristic peaks. Compared with the reference standards, 6 major chromatographic peaks were characterized and identified, with sum of peaks area over 70% according to area normalization method. Additionally, the similarity analysis and HCA analysis were performed and the results got mutual authentication. In quantitative analysis, the four compounds showed good regression(r>0.9995) within test ranges and the recovery of the method was in the range of 97.9%-103.7%. Conclusion The results revealed that the method of reference chromatographic fingerprints combined with multiple compounds determination could be used as an efficient strategy for systematic quality evaluation of Rhizoma Drynariae.
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Rhizoma Drynariaed is commonly used for orthopedics, and the chemical composition mainly were flavonoids, triterpenoids, and phenolic glycosides, etc.It has good bioactivity of healing, anti-osteoporosis, anti-inflammatory activity.This paper reviewed the research on chemical constituents and pharmacological activities of Rhizoma Drynariae at home and abroad in recent years, lay the foundation for the further research and development activity of Rhizoma Drynariae.
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Objective To observe the expression of Glu、mGluR 5 and EAAT 1 in bone tissues of ovariectomized osteoporotic rats and the effects of Total Flavonoids of Rhizoma Drynariae (TFRD) on it. Methods 45 SPF 3-month-old Sprague-Dawley (SD) female rats were randomly divided into sham operation (Sham, n=15) group and ovariectomized (OVX, n=30) group. The osteoporotic(OP) model was established by bilateral ovariectomy, 14 weeks later, we measured bone mineral density(BMD) by dual-energy X-ray and determined that OP model was successfully replicated, OVX group rats were then divided into OVX group (n=15) and OVX+TFRD group (n=15). The OVX+TFRD group was given TFRD for 12 weeks. Glutamate (Glu), metabotropic glutamate receptor 5 (mGluR 5), and Glutamate/Aspartate Transporter (GLAST/EAAT 1)’s expression of femur was examined in order to clarify the characteristics of bone glutamate signaling pathway and the effects of TFRD on it. Results Glu and ionotropic receptors mGluR 5 mainly distributed in bone marrow cells and osteoblasts closed to the bone marrow cavity walls. There were no significant differences in Glu expression among Sham group, OVX group and OVX+TFRD group. The mGluR 5 expression of OVX+TFRD group was significantly higher than that of Sham group and OVX group(P=0.009), while no significant difference was found between the latter two groups. In addition to large distribution in bone marrow cells, small amount of transporter EAAT 1 was noted to express in bone cells of the bone lacunae. There were no significant differences in EAAT 1 expression among the three groups. Conclusion In bone glutamate signaling pathway, this study demonstrated that TFRD could significantly improve the ionotropic receptor mGluR 5’s expression, but had no inlfuence for Glu and EAAT 1.
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AIM: To study the effects of different process methods on the content of total flavonoids and the aqueous extract from Rhizoma Drynariae. METHODS: The contents of total flavonoids of Rhizoma Drynariae processed in different ways were assayed by polyamide chromatography and ultraviolet spectroplotometry; the contents of the aqueous extract were assayed by extraction in water. RESULTS: The contents of total flavonoids and the aqueous extract in Rhizoma Drynariae increased after it was processed and purified. The contents of total flavonoids and the aqueous extract in Rhizoma Dyrnariae hotted in sands, at constant temperature oven and microwave process were not influenced, but easier to be extracted in water by these methods. CONCLUSION:Microwave processis better than the other methods.