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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Change of icariin content in five Epimedium species by heating process / 中国中药杂志

The change of icariin( ICA) content in thirty-three samples of five Epimedium species listed in the Chinese Pharmacopoeia( 2015 edition),including E. brevicornu,E. sagittatum,E. pubescens,E. koreanum,and E. wushanense has been investigated in this study. The results indicated that the optimized process procedure was baking at 150 ℃ for 30 min,and 3'''-carbonyl-2″-β-L-quinovosyl icariin( CQICA) could not be translated into ICA and ICA could be converted under this heating process condition. ICA increased remarkably after the heating process by 1-3 times in E. brevicornu,E. wushanense and E. koreanum,and increased lightly in E. brevicornum and E. pubescens,while ICA slightly increased or decreased in E. sagittatum and E. wushanense.

Fingerprint of Epimedium wushanense Based on Principal Component，Factor and Cluster Analysis / 中国实验方剂学杂志

Objective: To establish the fingerprint of Epimedium wushanense by HPLC,and study the comprehensive quality of E. wushanense by combining principal components,factors and cluster analysis,so as to provide theoretical basis for its quality evaluation. Method: The chromatographic column was Agilent infinitylab poroshell 120 SB-C18 (3.0 mm×100 mm,2.7 μm),the flow phase was acetonitrile (A)-water (B) with a gradient of 0-5 min,25%-26%A;5-6 min,26%-34%A;6-11 min,34%-38.5%A;11-17 min,38.5%-100%A;17-20 min,100%A,the flow rate was 0.8 mL·min- 1,the detection wavelength was 270 nm,and the column temperature was 30℃. Result: The cluster analysis better classified E. wushanense from different producing areas. E. wushanense from Guizhou province and E. wushanense from Chongqing were classified as class Ⅱ. E. wushanense from Guizhou province and Chongqing were far apart, indicating that the quality of E. wushanense varies from place to place affected by environment and climate. The results of principal component analysis showed that the quality of E. wushanense produced in Chongqing was better than that of E. wushanense produced in Guizhou. Among them,CQWS-02 (Yaque village,Guanyang town,Wushan county,Chongqing) and CQWS-10 (Hewan, Guanyang town,Wushan county,Chongqing) can be considered in the selection of high-quality varieties. In addition,No.1 common peak (epimedin A),No.2 common peak (epimedin B),No.4 common peak (icariin) and No.5 common peak (unknown component) in the fingerprint of the test samples could be used as the evaluation index components of E. wushanense quality. Conclusion: Principal components,factors and cluster analysis are used to achieve the rapid analysis, and their respective advantages are brought into full play for mutual verification and supplement. And the quality of E. wushanense in different origins can be comprehensively evaluated in all-round ways.

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.

Evaluation of metabolism-effect/toxicity of Epimedium sagittatum based on zebrafish M-Act/Tox integration method / 中草药

Objective To evaluate the metabolism, anti-osteoporosis efficacy and toxicity of Epimedium sagittatum based on zebrafish M-Act/Tox integration method. Methods The safety of E. sagittatum (crude drug 200, 500, 1 000, 1 500, and 2 000 μg/mL) was evaluated with 1-6 dpf zebrafish, the morphology of fish organs was observed and the number of deaths was counted and the half death concentration of zebrafish (LC50) was calculated; The 5 dpf zebrafish were exposed to E. sagittatum (crude drug 250 μg/mL ) for 24 h, six representative flavonoids, including epimedin A, epimedin B, epimedin C, icariin, sagittatoside C, and baohuoside I, were analyzed; The zebrafish osteoporosis model was induced with 25 μmol/L prednisolone, microscopic detection and digital imaging of zebrafish larvae of each group cultured to 8 dpf were performed using alizarin red, and the bone staining area was quantitatively analyzed by image software to evaluate the anti-osteoporosis activity of E. sagittatum. Results E. sagittatum caused zebrafish poisoning at crude drug 500 μg/mL and above concentration (organs deformation or death), and toxicity was related to drug concentration and exposing time; After the action of zebrafish, epimedin A, epimedin B, epimedin C, and icariin were almost completely transformed, and sagittatoside C and baohuoside I were the main metabolites; E. sagittatum had significant anti-prednisolone-induced bone loss in zebrafish at a certain concentration (crude drug 6.25, 12.5, and 25 μg/mL). Conclusion The metabolism, anti-osteoporosis efficacy and toxicity of E. sagittatum are evaluated using zebrafish. The zebrafish M-Act/Tox integration method integrates the advantages of zebrafish metabolism, osteoporosis model and toxicity evaluation method, and realizes the effect/toxicity evaluation based on in vivo process, providing new ideas and method for screening anti-osteoporosis Chinese medicine.

Optimization of extraction process of Shenqi Qiangxin Tablets based on pharmacodynamic test and analytic hierarchy process / 中草药

Objective: To optimize the extraction technology of Shenqi Qiangxin Tablets (SQT). Methods: With the improvement of heart lesion of pharmacological model of isoproterenol induced heart failure in rats as the index, pharmacological efficacy test was used to screen extracting conditions of the technology. The extraction technology was optimized by analytic hierarchy process combined with principal component analysis, single factor and orthogonal tests using each content of solid matter, ginsenosides Rg1, Re as indexes. And the verification test was carried out by using solid mass and icariin content as indexes. Results: Pharmacological efficacy test showed that technology 4 was superior. The optimal extraction condition of technology 4 was as follow: five medicinal materials including red ginseng and astragalus were reflux extracted three times with 50% ethanol, 11 fold for the first time, 10 fold for the second and three times, 2.5 h for each extraction; Epimedium and the other two medicinal materials were decocted three times with water, 19 fold for the first time, 16 fold for the second and third times, 1.5 h for each decction. The verification test showed that the average yield of ethanol extracted solids was 19.78%, and the average extraction rate of ginsenoside Rg1 and Re was 77.52%; The average value of water extracted solids was 16.58%, and the average extraction rate of epimedium was 90.98% (RSD < 2.0%, n = 3). Conclusion: The optimized extraction technology was stable and feasible.