Improvement of solubility of epimedium flavonoid secondary glycoside components by traditional Chinese medicine polysaccharides and its mechanism / 中国中药杂志

The poor solubility of insoluble components of traditional Chinese medicine(TCM) is an important factor restricting the development of its preparations. Natural polysaccharides of TCM can be used as functional components to increase the solubility of insoluble components. Epimedium flavonoid secondary glycoside components(EFSGC) have been shown to have positive effects on the prevention and treatment of osteoporosis, but they exhibit poor solubility. Therefore, the strategy of solubilizing EFSGC with TCM polysaccharides was adopted, and its effect on the permeability and stability of EFSGC was evaluated in this study. Based on the equilibrium solubility experiment of EFSGC, it was found that Panax notoginseng crude polysaccharide(PNCP) had the best solubilization effect on EFSGC among the ten kinds of TCM polysaccharides, which increased the solubility of EFSGC from 0.8 mg·mL~(-1) to 13.3 mg·mL~(-1). It should be noted that after the solubilization of EFSGC by preparation technology, the effects on permeability and stability should be considered. Therefore, this study also investigated these two properties. The results showed that PNCP increased the effective transmittance of EFSGC from 50.5% to 71.1%, which could increase the permeability of EFSGC significantly. At the same time, it could improve the stability of EFSGC in the simulated gastric juice environment. In order to explain the solubilization mechanism of PNCP on EGSGC, critical micelle concentration, particle size, potential, differential scanning calorimetry, and infrared spectroscopy were analyzed. It was preliminarily inferred that the mechanism was as follows: PNCP and EFSGC could self-assemble into aggregates for solubilization by intermolecular hydrogen bonding interaction in water. In summary, PNCP can not only improve the solubility of EFSGC but also improve its permeability and stability. This study lays the foundation for the application of TCM polysaccharides as a functional component to solubilize insoluble components.

Aqueous extract of Epimedium sagittatum mitigates pulmonary fibrosis in mice / 中国中药杂志

This study aims to investigate the intervention effect of the aqueous extract of Epimedium sagittatum Maxim on the mouse model of bleomycin(BLM)-induced pulmonary fibrosis, so as to provide data support for the clinical treatment of pulmonary fibrosis. Ninety male C57BL/6N mice were randomized into normal(n=10), model(BLM, n=20), pirfenidone(PFD, 270 mg·kg~(-1), n=15), and low-, medium-, and high-dose E. sagittatum extract(1.67 g·kg~(-1), n=15; 3.33 g·kg~(-1), n=15; 6.67 g·kg~(-1), n=15) groups. The model of pulmonary fibrosis was established by intratracheal instillation of BLM(5 mg·kg~(-1)) in the other five groups except the normal group, which was treated with an equal amount of normal saline. On the day following the modeling, each group was treated with the corresponding drug by gavage for 21 days. During this period, the survival rate of the mice was counted. After gavage, the lung index was calculated, and the morphology and collagen deposition of the lung tissue were observed by hematoxylin-eosin(HE) and Masson staining, respectively. The levels of reactive oxygen species(ROS) in lung cell suspensions were measured by flow cytometry. The levels of glutathione peroxidase(GSH-Px), total superoxide dismutase(T-SOD), and malondialdehyde(MDA) the in lung tissue were measured. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling(TUNEL) was employed to examine the apoptosis of lung tissue cells. The content of interleukin-6(IL-6), chemokine C-C motif ligand 2(CCL-2), matrix metalloproteinase-8(MMP-8), transforming growth factor-beta 1(TGF-β1), alpha-smooth muscle actin(α-SMA), E-cadherin, collagen Ⅰ, and fibronectin in the lung tissue was measured by enzyme-linked immunosorbent assay(ELISA). The expression levels of F4/80, Ly-6G, TGF-β1, and collagen Ⅰ in the lung tissue were determined by immunohistochemistry. The mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue were determined by qRT-PCR. The content of hydroxyproline(HYP) in the lung tissue was determined by alkaline hydrolysation. The expression of α-SMA and E-cadherin was detected by immunofluorescence, and the protein levels of α-SMA, vimentin, E-cadherin in the lung tissue were determined by Western blot. The results showed the aqueous extract of E. sagittatum increased the survival rate, decreased the lung index, alleviated the pathological injury, collagen deposition, and oxidative stress in the lung tissue, and reduced the apoptotic cells. Furthermore, the aqueous extract of E. sagittatum down-regulated the protein levels of F4/80 and Ly-6G and the mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue, reduced the content of IL-6, CCL-2, and MMP-8 in the alveolar lavage fluid. In addition, it lowered the levels of HYP, TGF-β1, α-SMA, collagen Ⅰ, fibronectin, and vimentin, and elevated the levels of E-cadherin in the lung tissue. The aqueous extract of E. sagittatum can inhibit collagen deposition, alleviate oxidative stress, and reduce inflammatory response by regulating the expression of the molecules associated with epithelial-mesenchymal transition, thus alleviating the symptoms of bleomycin-induced pulmonary fibrosis in mice.

Epimedium regulates stem cell-like character of breast cancer via miR-148a / 中国药理学通报

Aim To observe the effect of epimedium on the proliferation and stem cell-like character expression of breast cancer cells, and investigate the relationship between the inhibition of stem cell-like character and miR-148a by epimedium, and its molecular mechanism. Methods After treatment with different concentrations of epimedium, cell viability and population dependence were detected by MTT assay and colony formation assay; the breast cancer stem cell-derived mammosphere formation was examined by Mammosphere assay; the expression levels of CD44,ALDH-1, Oct4,BMIl and EpCAM were detected by qPCR; the protein expression levels of EpCAM, SOX4, ZO-1, E-cadherin and vimentin were detected by Western blot; the protein localization of EpCAM was observed by im-munofluorescence assay; the effect of epimedium on migration was detected by wound healing assay. The miR-148a mimic was transfected into MDA-MB-231 cells, and the effects of epimedium on stem-like character expression of transfected MDA-MB-231 cells were observed. Results Epimedium significantly inhibited the proliferation and population dependence of MDA-MB-231 cells (P < 0.05 ), and reduced the breast cancer stem cell-derived mammosphere formation; compared with control group, epimedium significantly decreased mRNA levels of CD44, ALDH-1, Oct4, BMI1 and EpCAM (P <0.05) ,decreased protein contents of EpCAM, SOX4 and Vimentin (P < 0.05 ), up-regulated the protein expression of ZO-1 and e-cadherin ( P <0.05) ,and decreased the migration ability of MDA-MB-231 cells (P < 0.05). Epimedium up-regulated the expression of miR-148a in MDA-MB-231 cells (P <0.01). YYH + miR-148a mimic group significantly inhibited stem-like character expression and EMT process of breast cancer MDA-MB-231 cells compared with control group (P <0.05). Conclusions Epimedium can inhibit the proliferation of MDA-MB-231 cells, which may be related to the up-regulation of miR-148a, decrease of stem-like character expression of breast cancer cells,and inhibition of EMT.

A new flavonoid glycoside from Epimedium sagittatum / 药学学报

Fourteen flavonoids were isolated and purified from Epimedium sagittatum by various chromatography techniques such as macroporous adsorbent resin, silica gel, ODS, Sephadex LH-20, HW-40C and semi-preparative HPLC. Their structures were identified by analysis of physicochemical properties and spectral data, and determined as 3′-hydroxy-baohuoside-Ⅱ (1), huazhongilexone-7-O-β-D-glucopyranoside (2), kaempferol-3-O-α-L-rhamnoside (3), baohuoside-Ⅱ (4), icariside-Ⅱ (5), kaempferol 3,7-di-O-α-L-rhamnopyranoside (6), (+)-aromadendrin (7), kaempferol 3-O-(2-O-β-D-apiofuranosyl)-α-L-rhamnopyranoside (8), sagittatoside A (9), 2″-O-rhamnosyl icariside-II (10), apigenin-7-O-β-D-glucoside (11), quercetin 3-O-β-D-apiofuranoyl-(1→2)-α-L-rhamnopyranoside (12), kaempferol (13), icariin (14). Among them, compound 1 is a new compound, while compounds 2, 6-8, 11, and 12 were isolated from E.sagittatum for the first time.

Analysis and evaluation of bioactive constituents from different parts of Epimedium brevicornum / 中国中药杂志

A comprehensive analytical method based on ultra-fast liquid chromatography coupled with triple quadrupole/linear ion trap tandem mass spectrometry(UFLC-QTRAP-MS/MS) was established for simultaneous determination of the content of 45 bioactive constituents including flavonoids, alkaloids, amino acids, phenolic acids, and nucleosides in Epimedium brevicornum. The multiple bioactive constituents in leaves, petioles, stems and rhizomes of E. brevicornum were analyzed. The gradient elution was performed at 30 ℃ in an XBridge~® C_(18) column(4.6 mm×100 mm, 3.5 μm) with 0.4% formic acid aqueous solution-acetonitrile as the mobile phase at a flow rate of 0.8 mL·min~(-1). Single factor experiment and response surface methodology were employed to optimize the extraction conditions. Multivariate statistical analyses including systematic cluster analysis(SCA), principal component analysis(PCA), partial least squares discriminant analysis(PLS-DA), and one-way analysis of variance(One-way ANOVA) were carried out to classify the samples from different parts and identify different constituents. Grey relation analysis(GRA) and entropy weight-TOPSIS analysis were performed to build a multi-index comprehensive evaluation model for different parts of E. brevicornum. The results showed that there was a good relationship between the mass concentrations of 45 constituents and the corresponding peak areas, with the correlation coefficients(r) not less than 0.999 0. The precision, repeatability, and stability of the established method were good for all the target constituents in this study, with the relative standard deviations(RSDs) less than 5.0%(0.62%-4.9%) and the average recovery of 94.51%-105.7%. The above results indicated that the bioactive constituents varied in different parts of E. brevicornum, and the overall quality followed the trend of leaves > petioles > rhizomes > stems. This study verified the rationality of the Chinese Pharmacopoeia(2020 edition) stipulating that the medicinal part of E. brevicornum is the leaf. Moreover, our study indicated that the rhizome had the potential for medicinal development. The established method was accurate and reliable, which can be used to comprehensive evaluate and control the quality of E. brevicornum. This study provides data reference for clarifying the medicinal parts and rationally utilizing the resources of E. brevicornum.

Taxonomy of Epimedium (Berberidaceae) with special reference to Chinese species / 中草药·英文版

Epimedii Herba is a commonly used traditional Chinese herbal medicine. Five Epimedium species are included in Chinese Pharmacopoeia and most species of Epimedium are used as Epimedii Herba in practical application. However, as the largest herbaceous genus of the Berberidaceae, Epimedium has many taxonomic controversies which hinder the effective use of Epimedii Herba. This paper reviewed the taxonomic research related to Epimedium, including taxonomic history, taxonomic values of morphological characters, species and distribution, infra-genera taxonomic system and the taxonomic research of Chinese Epimedium. For instance, we recognized Epimedium wushanense and clarified that the species, as described in Flora Reipublicae Popularis Sinicae and Flora of China, actually includes four Epimedium species similar in leaflet shape. In general, it was recognized here that Epimedium comprises 62 species, of which 52 species are distributed in China. For Chinese Epimedium species with the most taxonomic problems, the taxonomic research on the taxa was reviewed and the newest species key was proposed along with proposals for those taxonomic problems needing further resolution. This review is of great implication for the identification, exploration and utilization of Epimedii Herba.

New anti-pulmonary fibrosis prenylflavonoid glycosides from Epimedium koreanum / 中国天然药物

Four new prenylflavonoid glycosides, namely koreanoside H-K (1-4), together with eleven known ones (5-15) were isolated from the leaves of Epimedium koreanum Nakai. Their structures were elucidated by 1D NMR, 2D NMR, HR-ESI-MS, IR and UV. The identification of the sugar moieties was carried out by means of acid hydrolysis and HPLC analysis of their derivatives. It is worth noting that compound 3 and compound 4 were elucidated to contain fucose and quinovose moieties, which were two extremely rare sugar units from the genus Epimedium. The anti-pulmonary fibrosis activity of the new compounds was evaluated using A549 cell line. Compounds 1, 2 and 4 showed significant anti-pulmonary fibrosis activities.

Herba Epimedii's biotransformation in vitro simulated gastrointestinal digestion and faecal fermentation systems / 中国药理学与毒理学杂志

OBJECTIVE Epimedium is rich in a variety of beneficial active ingredients, and has been widely used in the ethnopharmacological practices, however, its biotransformation in gastrointestinal digestions remain unclear. This study aimed to investigate the dynamic changes of components and biological activity of Epimedium in the in vitro simu? lated digestion and subsequent human faecal fermentation. METHODS The models of in vitro simulated saliva, gastric and intestinal digestion, as well as colonic fermentation were constructed to simulate the digestion process of Epimedium. The dynamic changes of components of Epimedium during the simulated digestions in vitro and subsequent human faecal fermentation were investigated by UPLC-MS, HPLC-DAD combined with principal component analysis (PCA) and multi-ingredient quantitative analysis. RESULTS A variety of metabolites with high contents were produced after 0.5 h of intestinal digestion and colonic fermentation 0.5 h. Application of PCA to HPLC data showed the obvious separation of colonic fermentation 0.5 h stage samples from other colonic fermentation stages samples (24, 48 and 72 h). Addition? ally, non-digestion and saliva digestion stage samples clustered together, and there was obvious separation between intestinal digestion samples and gastric digestion samples. The contents of epimedium C, icariin and baohuside I all increased significantly after intestinal digestion [58.70 ± 7.08, 47.15 ± 5.68 and (12.78 ± 0.55) mg · g-1] compared with gastric digestion [29.00 ± 5.65, 17.40 ± 4.55 and (2.77 ± 0.19) mg·g-1]. There were significant differences between sample after 0.5 h of colonic fermentation [64.22 ± 9.32, 51.26 ± 6.33 and (16.68 ± 3.19) mg·g-1] and other time points (24, 48 and 72 h) in components and the contents of active ingredient, and the content of these components all decreased with the fermentation time. The ability of scavenging ABTS free radicals [IC50=(0.29 ± 0.02) g · L-1] increased significantly compared with gastric digestion [(1.57 ± 0.02) g·L-1], and after 0.5 h of colonic fermentation, the ability also increased significantly. CONCLUSION Gastrointestinal digestion had a significant impact on the contents of active components in Epimedium, and the metabolism of these components mainly occurred in the colon. The intestinal digestion and colonic fermentation significantly improved the anti-ABTS activity of epimedium.

Effects and mechanism of epimedium polysaccharide on solubility of icariin and baohuoside Ⅰ / 中国中药杂志

This study evaluated the effects of epimedium polysaccharide(EPS) on the solubility of icariin and baohuoside Ⅰ so as to preliminary explore its solubilization function and the underlying mechanism. The solubility of these two insoluble flavonoids in water and polysaccharide solutions was compared by high performance liquid chromatography, and the mechanism was investigated by diffe-rential scanning calorimetry(DSC) and critical micelle concentration determination. The results indicated that their solubilization in crude EPS solutions was concentration-dependent. The solubility of icariin and baohuoside Ⅰ in 20 mg·mL~(-1) EPS-1-1 was 9.05 times and 5.76 times that in water, respectively; while their solubility in 20 mg·mL~(-1) EPS-2-1 was 10.55 and 8.39 times that in water, respectively. The change of the DSC thermograms suggested the formation of new complexes from icariin and baohuoside Ⅰ with polysaccharides. The critical micelle concentrations proved the micellar properties of both EPS-1-1 and EPS-2-1. In short, EPS can significantly increase the solubility of icariin and baohuoside Ⅰ, the mechanism of which may be related to the formation of micellar complexes between EPS and insoluble flavonoids.

Establishment of HPLC Fingerprint ，Multivariate Statistical Analysis and Processing Technology of Different Suet Oil-baked Epimedium brevicornum / 中国药房

OBJECTIVE：To establish HPLC fingerprint of different products of suet oil-baked Epimedium brevicornum ，and to screen the optimal baking technology. METHODS ：HPLC method was adopted. Using icariin as reference ，HPLC fingerprints of 22 batches of samples were drawn. The similarity was evaluated by using Similarity Evaluation System of TCM Chromatographic Fingerprint（2012 edition），and common peaks were confirmed. HCA ，PCA and OPLS-DA analysis were performed by SIMCA 14.1 statistical software. Taking variable importance in the project ＞1 as criteria ，biomarkers affecting the quality difference of suet oil-baked E. brevicornum were screened ；using mass marker as index ，the baking technology was screened by baking technology. RESULTS：There were 18 common peaks in 22 batches of samples. The similarities were between 0.831 and 0.991. Totally 7 common peaks were identified ，i.e. epimedin A ，epimedin B ，epimedin C ，icariin，sagittatoside A ，sagittatoside B ，baohuoside Ⅰ. The 22 batches of samples were clustered into two categories ，S19-S22 were clustered into category Ⅰ and S 1-S18 were clustered into category Ⅱ. The category Ⅱ was sub-clustered into category Ⅱa（S15-S18），category Ⅱb（S10-S14），category Ⅱc （S1-S9）；the result of PCA analysis was consistent with above results. OPLS-DA showed that the biomarkers affecting the quality difference were icariin ，sagittatoside B and baohuoside Ⅰ. The results of kinetic studies showed that the content of icariin when baked at 180 ℃ for 25 min or 190 ℃ for 20 min，that of baohuoside Ⅰ when baked at 180 ℃ for 30 min or 190 ℃ for 15 min and that of epimedin B when baked at 210 ℃ for 18 min were the highest ；according to above results ，the optimal baking technology was baking at 180 ℃ for 25-30 min or 190 ℃ for 15-20 min. CONCLUSIONS ：Established fingerprint is stable ， reliable and reproducible. The multivariate statistical analysis can be used for the changes of chemical components in E. brevicornum under different baking condition and preliminary selection of baking technology.

Evaluation of cultivated and wild Herba Epimedii using UHPLC-PDA-Q-TOF/MSE / 药学学报

Qualitative and relatively quantitative methods were used to study the quality of cultivated Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium myrianthum Stearn, and Epimedium pubescens Maxim by ultra high-performance liquid chromatography coupled with photo-diode array and quadrupole time-of-flight mass spectrometry (UHPLC-PDA-Q-TOF/MSE). Thirty-two compounds in cultivated and wild samples of E. sagittatum, E. myrianthum and E. pubescens were identified using UHPLC-Q-TOF/MSE combined with the UNIFI data analysis platform. Principal component analysis (PCA) was used to compare the cultivated and wild samples of these three species. The results show that the chemical compositions of cultivated samples were consistent with the corresponding wild samples. UHPLC-PDA was used to determine the relative content of 12 flavonoids as well as total flavonoids in all samples. The results show that the relative chemical content of these flavonoids in cultivated and wild samples is similar and the quality of cultivated Epimedium is more stable. These qualitative and relatively quantitative methods using UHPLC-PDA-Q-TOF/MSE combined with the UNIFI data analysis platform and PCA can be used to study the quality of cultivated Herba Epimedii. This research provides a scientific basis for the cultivation and rational development and utilization of Epimedium medicinal materials.

Study on the Effect and Mechanism of Total Flavonoids of Epimedium brevicornu on Postmenopausal Osteoporosis Model Rats Based on BMP/Runx 2/Osx Signaling Pathway / 中国药房

OBJECTIVE：To study the effect of total flavonoids of Epimedium brevicornu on postmenopausal osteoporosis （PMOP）model rats based on BMP/Runx 2/Osx signaling pathway so as to confirm the mechanism of preventing and treating osteoporosis（OP）. METHODS ：By body mass stratification ，50 rats were randomly divided into sham operation group ，model group，E. brevicornu total flavonoids low-dose and high-dose groups [265，530 mg/（kg·d）]，estradiol group [0.09 mg/（kg·d）]， with 10 rats in each group. Except that sham operation group underwent sham operation ，PMOP model was established by ovariectomy and castration in other groups. After modeling ，they were given normal diet for 2 months and then given relevant medicine intragastrically for consecutive 84 d，once a day ；rats in sham operation group and model group were given equal volume of normal saline. After last medication ，bone mineral density （BMD）of femur and vertebrae of the right lower limb ，the number of trabecular bone （Tb.N），trabecular bone thickness （Tb.Th）and trabecular separation （Tb.Sp）of femur were determined in each group. The serum levels of Ca 2+，OC and P 1NP in serum were detected by ELISA. HE staining was used to observe pathological changes of femur. mRNA and protein expressions of BMP ，Runx2 and OSX in bone tissue were detected by RT-PCR and Western blotting. RESULTS ：Compared with sham operation group ，BMD of femur and vertebrae ，serum levels of Ca 2 +，OC and P 1NP， Tb.N and Tb.Th of femur ，mRNA and protein expression of BMP ，Runx2 and Osx in femur were decreased significantly in model group，while Tb.Sp of femur was increased significantly （P＜0.01）；the structure of trabecular bone was disordered and the fracture was obvious. Compared with model group ，above indexes of rats in administration groups were improved significantly （P＜ 0.05 or P＜0.01），and the effect of E. brevicornu total flavonoids was dose-dependent （P＜0.05）；the number of trabecular bone increased， arranged orderly and the structure was more brevicornu total flavonoids can improve OP ，the mechanism of which may be associated with com promoting the activity of BMP/Runx 2/Osx signaling pathway.

Effects of light quality on growth and icariin flavonoid content of Epimedium pseudowushanense under different light intensity / 中国中药杂志

In this study, the growth index including plant height, compound leaf area, specific leaf area, leaf water content, number of branches, and leaf biomass per plant and the icariin flavonoids such as epimedin A, epimedin B, epimedin C and icariin of Epimedium pseudowushanense were determined on 30 d and 60 d under light intensity(18.2±2.5) μmol·m~(-2)·s~(-1)(L1) and(90.9 ±2.5) μmol·m~(-2)·s~(-1)(L2), and white light as control, red light, blue light and yellow light were used as three light quality treatments, to study the effect of light quality on the growth and flavonoids accumulation of E. pseudowushanense. The E. pseudowushanense was sui-table for growth under L1 light intensity, the blue light treatment significantly reduced the leaf area, but had little effect on the stem height, the red light treatment and the yellow light treatment had no obvious effect on the stem height and leaf area, but the yellow light treatment significantly increased the germination of new branches, and had a sustained promoting effect, and the biomass was significantly higher than the white light treatment at 60 d. The content of icariin flavonoids in red light, blue light and yellow light treatment was higher than that in white light treatment at 30 d and 60 d under L1 light intensity, while yellow light treatment promoted the synthesis of icariin flavonoids to the largest extent, which was 1.8 and 1.9 times of white light treatment(30 d and 60 d).Under L2 light intensity, the effect of strong light on promoting stem germination became the main factor, while the yellow light treatment showed no significant effect on promoting stem germination, and the red light treatment exhibited a significant effect on reducing leaf area. Icariin flavonoids under red light, blue light and yellow light treatment were all lower than that under white light treatment, that is, the effect of white light treatment on the synthesis of icariin flavonoids is better than red light, blue light and yellow light treatment. When the time of strong light treatment was longer, the degradation range of icariin flavonoids in other light treatment appeared, while red light treatment promotes the synthesis of icariin flavonoids. Therefore, the influence of light quality on E. pseudowushanense is quite different under different light intensity, no matter from growth index or flavonoid content index. The results support that the biomass and icariin flavonoid content can be increased by providing appropriate red and yellow light.

Effect of glycoside of short-horned epimedium Herb on the expressions of transforming growth factor β 1 and basic fibroblast growth factor induced membrane by Masquelet technology / 中国修复重建外科杂志

Objective: To observe the expressions of transforming growth factor β 1 (TGF-β 1) and basic fibroblast growth factor (bFGF) induced membrane by Masquelet technique in rats treated with glycoside of short-horned epimedium Herb, and to explore the effect of glycoside of short-horned epimedium Herb on Masquelet induced membrane. Methods: Sixty 3-month-old male Wistar rats were randomly divided into 3 groups with 20 rats in each group; a tibial bone defect (6 mm in length) model was established. The blank group (group A) was not treated; the control group (group B) and the experimental group (group C) were filled with vancomycin antibiotic bone cement in the drawing stage, and the bone cement was completely solidified. Group C was given perfused flavonoids glycoside of short-horned epimedium Herb (10 μmol/L) by gavage once a day (0.3 mL) from 1 day after operation, and groups A and B were given the same amount of normal saline by gavage. After operation, the recovery and wound healing of experimental animals were observed; at 4 weeks after operation, X-ray film was taken to observe the recovery of bone defect of proximal tibia; at 6 weeks after operation, the bone defect was observed, and the morphological changes and vascularization degree of granulation tissue and induction membrane tissue were observed; the expressions of TGF-β 1 and bFGF were observed by immunohistochemistry staining and ELISA detection. Results: The bone defect models of the 3 groups were established successfully, and there was no abnormality after operation. The incisions healed by first intention after operation. At 4 weeks after operation, X-ray films of proximal tibial defect showed that there was obvious space in group A, while bone cement was filled and Kirschner wire fixation was good in groups B and C. At 6 weeks after operation, the gross observation showed that the granulation tissue was filled in the defect area in group A; transparent membrane was formed in groups B and C, and microvessels were seen in some areas, and the microvessels in group C were significantly more than those in group B. Immunohistochemical staining showed that the expressions of TGF-β 1 and bFGF were negative in group A, but they were expressed in groups B and C, and the expressions of TGF-β 1 and bFGF in group B were significantly less than those in group C. ELISA detection showed that the expressions of TGF-β 1 and bFGF in group C were significantly higher than those in groups A and B ( P0.05). Conclusion: Glycoside of short-horned epimedium Herb can significantly increase the expressions of TGF-β 1 and bFGF, accelerate the process of osteogenesis, and contribute to bone shaping and reconstruction.

Mechanisms of epimedium in the treatment of osteoarthritis based on network pharmacology and protein module / 中国组织工程研究

BACKGROUND: Pharmacological studies in modern Chinese medicine have shown that icariin has a very positive effect on osteoarthritis. Because of the complex chemical composition of Epimedium and its unclear mechanism underlying the treatment of osteoarthritis at the molecular level, network pharmacology is introduced to explain the potential chemical components and molecular mechanism of Epimedium in the treatment of osteoarthritis. This can provide a theoretical basis for future drug development and disease treatment. OBJECTIVE: To explore the molecular mechanism of Epimedium in the treatment of osteoarthritis based on network pharmacology. METHODS: TCMSP database was used to screen the active ingredients of Epimedium. TCMSP, Swiss Target Prediction and STITCH database were used to predict the regulatory targets of the active ingredients of Epimedium. OMIM, GeneCards and TTD database were used to predict the therapeutic targets of osteoporosis. The therapeutic target of Epimedium for osteoporosis was obtained by intersecting the therapeutic target of Epimedium and osteoarthritis. A drug-component-target-disease network was then constructed. The protein interaction was analyzed by STRING database, and the related signaling pathways and functions of protein modules were analyzed by DAVID database. RESULTS AND CONCLUSION: Twenty-three pharmacodynamic components of Epimedium were screened and 230 pharmacodynamic targets of Epimedium and 1 221 therapeutic targets of osteoarthritis were predicted. After crossing, 95 therapeutic targets of Epimedium for osteoporosis were obtained. Protein interaction analysis indicated that JUN, AKT1, RELA, MAPK1, IL6, CXCL8, MAPK8, MAPK14, FOS and IL1B were the core targets of protein interaction network. Key protein modules were mainly involved in interleukin receptor pathway, tumor necrosis factor signaling pathway, Toll-like receptor signaling pathway, T cell receptor signaling pathway, nuclear factor-kappa B signaling pathway and osteoclast differentiation pathway. They might play a role in the treatment of osteoarthritis by regulating many biological processes such as cell proliferation and apoptosis, immune cells and immune response, inflammatory factors and inflammatory response, and lipopolysaccharide cell response.

Distribution of Epimedium sagittatum and its research progress on quality characteristics / 中草药

To promote the rational development and utilization of Epimedium sagittatum, and to propose the utilization strategies and future research suggestions. The distribution was illuminated based on the examination of herbarium specimens and our field investigation. At the same time, the quality characteristics of different harvest time, different parts and different producing areas were summarized according to the literature review and our previous researches. In terms of the distribution, E. sagittatum is a widely distributed species in Epimedium. However, there were many errors in the identification of herbarium specimens. At least 13 taxa were wrongly identified as E. sagittatum. The distribution of E. sagittatum was not as extensive as previously recorded in the literature. Although the distribution of E. sagittatum has been recorded in 12 provinces and in Chongqing Municipality, it was mainly distributed in Anhui, Jiangxi, Hunan and Hubei provinces, and the resources in each region were not abundant. In terms of quality characteristics, there were great differences in both composition and contents of the active components among different germplasm. Some of the germplasm in Hunan, Hubei and Sichuan provinces were excellent. However, the stability and homogeneity were not ideal. Therefore, the utilization of E. sagittatum should depend on the quality characteristics. It was found that there were great differences among populations and which were closely related to the genetic basis. The effect of physiological ecology on quality and its mechanism, as well as the molecular mechanism of quality formation should be paid more attention in the future.

Research progress on the therapeutic effect of traditional Chinese medicine on fluorosis / 口腔疾病防治

@#Fluoride is an important trace element in the human body and has active chemical properties, but its safety range is narrow. Excessive intake of fluoride can easily lead to fluorosis and various metabolic disorders, mainly manifested as dental fluorosis and skeletal fluorosis. In recent years, an increasing number of scholars have studied traditional Chinese medicine (TCM) because of its long history, few side effects, convenient oral administration and abundant resources. This paper reviews the current research. The results of the literature review show that traditional Chinese medicine, such as asparagus, cistanche deserticola and quercetin, can effectively prevent and treat fluorosis diseases that affected soft and hard tissues of the body due to is antioxidant properties. Moreover, the protective effect of TCM on fluoride-excreting organs of the body can further restore the fluoride-excreting ability of the body and reduce the damage caused by excessive fluoride on tissues as well as reduce fluoride in tissues by combining with excessive fluoride in the body. TCM can effectively improve the pain and joint dysfunction caused by skeletal fluorosis. Traditional Chinese medicine has important value in the prevention and treatment of fluorosis by enhancing immunity, promoting metabolism, eliminating fluoride in vivo, and improving the self-regulation mechanism to prevent fluoride toxic damage in the body.

Content Determination of 6 Flavonoids in Epimedium brevicornu from Shenqi Yanshen Granules Based on HPLC-QAMS / 中国药房

OBJECTIVE： To establish a HPLC method for simultaneous determination of 6 flavonoids in Epimedium brevicornu from Shenqi yanshen granules， such as epimedin A1， epimedin A， epimedin B， epimedin C， icariin and baohuoside Ⅰ. METHODS： HPLC method was adopted. The determination was performed on Waters Symmetry C18 column with mobile phase consisted of acetonitrile-water （gradient elution） at the flow rate of 1.0 mL/min； the column temperature was 25 ℃， and detection wavelength was 270 nm. The sample size was 10 μL. Relative correction factors （fk/s） of each component to icariin （reference substance） were established by multi-point correction method and slope correction method on the basis of external standard method to calculate the contents of each component. The contents of 6 flavonoids in 4 batches of Shenqi yanshen granules determined by HPLC external standard method were compared with by multi-point correction method and slope correction method. Feasibility and accuracy of quantitative analysis of multi-components by single-marker （QAMS） were validated. RESULTS： The linear range of epimedin A1， epimedin A， epimedin B， epimedin C， icariin and baohuoside Ⅰ were 2.03-50.80 μg/mL （r＝0.999 5）， 4.34-108.60 μg/mL （r＝0.999 5）， 2.26-56.40 μg/mL （r＝0.999 5）， 4.14-103.60 μg/mL （r＝0.999 5）， 4.24-106.00 μg/mL （r＝0.999 5）， 1.78-44.60 μg/mL （r＝0.999 5）， respectively， the limits of detection were 65.80， 71.49， 74.26， 68.79， 70.56， 86.09 ng/mL， respectively； the limits of quantification were 196.62， 213.63， 223.72， 208.46， 215.96， 255.88 ng/mL， respectively； RSDs of precision， stability （24 h）， reproducibility tests were less than 2％ （n＝6）， respectively. The average recoveries were 96.03％-99.04％ （RSDs were 0.65％-1.04％， n＝6）. By multi-point correction method， fk/s of epimedin A1， epimedin A， epimedin B， epimedin C and baohuoside Ⅰ were 0.837， 0.818， 0.845， 0.831， 1.387， respectively； by slope correction method， fk/s of them were 0.835， 0.815， 0.851， 0.829， 1.419， respectively. There was no significant difference in content determination results between two correction methods of QAMS and external standard （P＞0.05）. CONCLUSIONS： Established HPLC- QAMS method is accurate and suitable for the quality control of epimedin A1， epimedin A， epimedin B， epimedin C， icariin and baohuoside Ⅰ in E. brevicornu from Shenqi yanshen granules.

Effect of Epimedium flavonoids on decreasing secretion function of Sertoli cells in testis of natural aging rats / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of total flavonoids of Epimedium (TFE) on the decline of secretion function of senile testis-supporting cells (Sertoli cells), and to explore their molecular mechanism from inositol-requiring enzyme 1α(IRE1α)/X-box- binding protein 1(XBP1) signaling pathway. METHODS Thirty-six SPF 18-month-old SD male rats were randomly divided into natural aging group, low and high dose TFE groups, with 12 rats in each group. Another ten 2-month-old rats were used as a youth control group. TFE was ig given once a day, and the ig administration was continued every five days after a two-day interval, and the administration lasted for 4 months. Then, the testicular index was calculated. The testicular histomorphology was observed by HE staining; while quantitative-PCR (qPCR) and Western blotting were used to detect the mRNA and protein expression levels of glial cell derived neurotrophic factor (GDNF), bone morphogenetic protein 4 (BMP4) and stem cell factor (SCF), and Western blotting protein expression levels of p-IRE1α and XBP1 in testes. The number of Sertoli cells in the testis and the localization of p-IRE1α in the testis were detected by immunofluorescence. RESULTS Compared with the young control group, the testicular index decreased significantly in the natural aging group (P<0.01), but was significantly up-regulated after TFE 40 m g · k g- 1 (P<0.05). The results of HE showed that the morphological structure of the testicular seminiferous tubules in the natural aging group were disorderly, and some spermatogenic cells were shed. The TFE group could improve the morphological structure of the seminiferous tubules of the testis. qPCR and Western blotting results showed that compared with the young control group, the mRNA and protein expression levels of GDNF, BMP4 and SCF in Sertoli cells in the natural aging group were significantly down-regulated (P< 0.01). However, the expression of secretory factors in TFE group was significantly up-regulated (P< 0.05, P<0.01). Western blotting results showed that compared with the young control group, the expression of p-IRE1α and XBP1 protein in the testis of the natural aging group was significantly down-regulated (P<0.01), but the expression of p-IRE1α and XBP1 protein in the TFE group was significantly up-regulated (P<0.01). The results of immunofluorescence also showed that there was no significant difference in the number of Sertoli cells between the natural aging group and the TFE group compared with the young control group, while the expression of p-IRE1α was localized both in cytoplasm of Sertoli cells and spermatogenic cells. CONCLUSION TFE can significantly improve the decline of secretion function of testicular Sertoli cells induced by aging, and the mechanism may be related to the regulation of IRE1α/XBP1 signaling pathway.

Effects of active components of Epimedium, Astragalus and Radix Puerariae on expression of hepcidin in hippocampus CA3 in APPswe/PSldE9 double transgenic Alzheimer's disease model mice / 中国药理学通报

Aim To explore the effect of a combination of Astragalus, Epimedium and Pueraria on the expression of hepcidin in hippocampal CA3 region of APPswe/PSldE9 double transgenic AD model mice. Methods Thirty 10-month-old APPswe/PSldE9 double transgenic model mice were randomly divided into three groups; the compound group, the model group and the deferox (D F X) group, and ten 10-month-old C57BL/6J mice were as normal control group. Subsequently, the brain tissue of the mice was taken out, and the expression of hepcidin in the hippocampal CA3 region of each group of mice was detected by immunofluorescence combined with Real-time PCR and Western blot. Results Compared withnormal group, the mRNA expression of hepcidin was down-regulated inmodel group. Afterintragastric administrationof active components of Epimedium, Astragalus and Radix Puerariae and DFX, hepcidin expression levels increased significantly compared with those in APPswe/PSldE9 double transgenic AD model mice hippocampal CA3 area (P 0. 05). Conclusions The effective components of Astragalus, Epimedium and Radix Puerariae can up-regulate the expression of hepcidin, It is suggested that the effective components of Epimedium, Astragalus, and Radix Puerariae have important theoretical significance in the clinical treatment of Alzheimer's disease with iron overload.