Efficient preparation of icariin from epimedin C by recyclable biphasic enzymatic hydrolysis.

Icariin is the most bioactive ingredient of Epimedium L. and a quality marker of Herba Epimedii. Conventional methods for production of Icariin are known to be inefficient, resulting in low yields and significant environmental pollution. This study aimed to develop a sustainable and effective biphasic enzymatic hydrolysis system for the efficient conversion of epimedin C to icariin. The biphasic system was created using butyl acetate and phosphate buffer (pH 4.5) at a ratio of 3:1 (V/V) along with α-L-rhamnosidase/epimedin C (2 U/1 mg) at 50 °C for 12 h. Consequently, 98.21% of epimedin C was hydrolysed to icariin, with 95.62% of the product being transferred to the organic phase. Even after four cycles of use, the conversion ratio remained high at 75.28%. Furthermore, this novel strategy was also used for the conversion of Epimedium brevicornu Maxim. extracts. The biphasic system represents a sustainable and effective method for icariin production, offering potential benefits for industrial applications.

Construction of the first high-density genetic linkage map and QTL mapping of flavonoid and leaf-size related traits in Epimedium.

BACKGROUND: Leaves are the main medicinal organ in Epimedium herbs, and leaf flavonoid content is an important criterion of Epimedium herbs. However, the underlying genes that regulate leaf size and flavonoid content are unclear, which limits the use of breeding for Epimedium development. This study focuses on QTL mapping of flavonoid and leaf-size related traits in Epimedium. RESULTS: We constructed the first high-density genetic map (HDGM) using 109 F1 hybrids of Epimedium leptorrhizum and Epimedium sagittatum over three years (2019-2021). Using 5,271 single nucleotide polymorphism (SNP) markers, an HDGM with an overall distance of 2,366.07 cM and a mean gap of 0.612 cM was generated by utilizing genotyping by sequencing (GBS) technology. Every year for three years, 46 stable quantitative trait loci (QTLs) for leaf size and flavonoid contents were discovered, including 31 stable loci for Epimedin C (EC), one stable locus for total flavone content (TFC), 12 stable loci for leaf length (LL), and two stable loci for leaf area (LA). For flavonoid content and leaf size, the phenotypic variance explained for these loci varied between 4.00 and 16.80% and 14.95 and 17.34%, respectively. CONCLUSIONS: Forty-six stable QTLs for leaf size and flavonoid content traits were repeatedly detected over three years. The HDGM and stable QTLs are laying the basis for breeding and gene investigation in Epimedium and will contribute to accelerating the identification of desirable genotypes for Epimedium breeding.

Epimedin C protects dexamethasone-induced osteoblasts through NRF1/RhoA pathway.

Osteoporosis (OP) is a metabolic bone disease that leads to decrease of bone strength and increase bone brittle and fracture. Dexamethasone (DXMS) usage is a common risk factor of OP. In present study, we found that the Epimedin C protect the DXMS-induced OP, Ras Homolog Family Member A transforming protein (RhoA) was increased in osteoblasts (OBs) and OP models. We further revealed that Nrf1 is a transcription factor that responds to Epimedin C and DXMS in modulating RhoA promoter. The results collectively demonstrate that Epimedin C functions as a positive modifier of RhoA via alteration of Nrf1 transcriptional activity on RhoA promoter, thereby, protecting OBs against OP. Our work is the first study identifying the Epimedin C function in balancing the OBs in OP model via Nrf1-RhoA.

Systematic Quality Evaluation of Epimedium wushanense T. S. Ying Based on Two Quality Control Standards: Total Flavonoid Glycosides and Epimedin C.

Two quality control standards, total flavonoid glycosides of Epimedii Folium and epimedin C of Epimedii Wushanensis Folium, were used to systematically evaluate the quality of Epimedium wushanense T. S. Ying, so as to provide reference for its germplasm screening and resource utilization. Seven representative populations of E. wushanense covering its main distribution areas were uniformly sampled during the flowering period. There were significant quality differences among the populations of E. wushanense. According to the quality standard of total flavonoid glycosides, all populations were superior to the quality standard of the Chinese Pharmacopoeia for Epimedii Folium, with more than 1.5 % total flavonoid glycosides. The variation ranges of epimedin A, epimedin B, epimedin C, icariin and total flavonoid glycosides were 0.40-0.76 %, 0.51-0.83 %, 1.70-9.31 %, 0.40-1.23 % and 3.05-10.61 %, respectively. According to the quality standard of epimedin C, all populations were better than the quality standard of the Chinese Pharmacopoeia for Epimedii Wushanensis Folium, with more than 1.0 % epimedin C. The variation range of epimedin C was 2.22-10.06 %. When comparing the results of the two methods, a trend of slightly lower mean values was found for total flavonoid glycosides, except for the HBXW population. The quality of E. wushanense was superior to both the quality standard of Epimedii Folium and Epimedii Wushanense Folium in the Chinese Pharmacopoeia. Epimedin C was the most abundant component. Among the investigated populations, HBXW and HBGK exhibited the highest quality, and may provide excellent genetic resources for standardized cultivation. In addition, the habitat of these populations can also serve a reference for cultivation conditions.

The Mechanism Underlying the Hypoglycemic Effect of Epimedin C on Mice with Type 2 Diabetes Mellitus Based on Proteomic Analysis.

Type 2 diabetes mellitus (T2DM) has become a worldwide public health problem. Epimedin C is considered one of the most important flavonoids in Epimedium, a famous edible herb in China and Southeast Asia that is traditionally used in herbal medicine to treat diabetes. In the present study, the therapeutic potential of epimedin C against T2DM was ascertained using a mouse model, and the mechanism underlying the hypoglycemic activity of epimedin C was explored using a label-free proteomic technique for the first time. Levels of fasting blood glucose (FBG), homeostasis model assessment of insulin resistance (HOMA-IR), and oral glucose tolerance, as well as contents of malondialdehyde (MDA) and low-density lipoprotein cholesterol (LDL-C) in the 30 mg·kg-1 epimedin C group (EC30 group), were significantly lower than those in the model control group (MC group) (p < 0.05), while the contents of hepatic glycogen, insulin, and high-density lipoprotein cholesterol (HDL-C), as well as activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the EC30 group were notably higher than those in the MC group (p < 0.05). The structures of liver cells and tissues were greatly destroyed in the MC group, whereas the structures of cells and tissues were basically complete in the EC30 group, which were similar to those in the normal control group (NC group). A total of 92 differentially expressed proteins (DEPs) were enriched in the gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In the EC30 vs. MC groups, the expression level of cytosolic phosphoenolpyruvate carboxykinase (Pck1) was down-regulated, while the expression levels of group XIIB secretory phospholipase A2-like protein (Pla2g12b), apolipoprotein B-100 (Apob), and cytochrome P450 4A14 (Cyp4a14) were up-regulated. According to the KEGG pathway assay, Pck1 participated in the gluconeogenesis and insulin signaling pathways, and Pla2g12b, Apob, and Cyp4a14 were the key proteins in the fat digestion and fatty acid degradation pathways. Pck1, Pla2g12b, Apob, and Cyp4a14 seemed to play important roles in the prevention and treatment of T2DM. In summary, epimedin C inhibited Pck1 expression to maintain FBG at a relatively stable level, promoted Pla2g12b, Apob, and Cyp4a14 expressions to alleviate liver lipotoxicity, and protected liver tissues and cells from oxidant stress possibly by its phenolic hydroxyl groups.

Epimedin C Alleviates Glucocorticoid-Induced Suppression of Osteogenic Differentiation by Modulating PI3K/AKT/RUNX2 Signaling Pathway.

Secondary osteoporosis is triggered mostly by glucocorticoid (GC) therapy. Dexamethasone (DEX) was reported to inhibit osteogenic differentiation in zebrafish larvae and MC3T3-E1 cells in prior research. In this research, we primarily examined the protective impacts of epimedin C on the osteogenic inhibition impact of MC3T3-E1 cells and zebrafish larvae mediated by DEX. The findings illustrated no apparent toxicity for MC3T3-E1 cells after administering epimedin C at increasing dosages from 1 to 60 µM and no remarkable proliferation in MC3T3-E1 cells treated using DEX. In MC3T3-E1 cells that had been treated using DEX, we discovered that epimedin C enhanced alkaline phosphatase activities and mineralization. Epimedin C could substantially enhance the protein expression of osterix (OSX), Runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALPL) in MC3T3-E1 cells subjected to DEX treatment. Additionally, epimedin C stimulated PI3K and AKT signaling pathways in MC3T3-E1 cells that had been treated using DEX. Furthermore, in a zebrafish larvae model, epimedin C was shown to enhance bone mineralization in DEX-mediated bone impairment. We also found that epimedin C enhanced ALPL activity and mineralization in MC3T3-E1 cells treated using DEX, which may be reversed by PI3K inhibitor (LY294002). LY294002 can also reverse the protective impact of epimedin C on DEX-mediated bone impairment in zebrafish larval. These findings suggested that epimedin C alleviated the suppressive impact of DEX on the osteogenesis of zebrafish larval and MC3T3-E1 cells via triggering the PI3K and AKT signaling pathways. Epimedin C has significant potential in the development of innovative drugs for the treatment of glucocorticoid-mediated osteoporosis.

Purification and Characterization of a Novel α-L-Rhamnosidase from Papiliotrema laurentii ZJU-L07 and Its Application in Production of Icariin from Epimedin C.

Icariin is the most effective bioactive compound in Herba Epimedii. To enhance the content of icariin in the epimedium water extract, a novel strain, Papiliotrema laurentii ZJU-L07, producing an intracellular α-L-rhamnosidase was isolated from the soil and mutagenized. The specific activity of α-L-rhamnosidase was 29.89 U·mg-1 through purification, and the molecular mass of the enzyme was 100 kDa, as assayed by SDS-PAGE. The characterization of the purified enzyme was determined. The optimal temperature and pH were 55 °C and 7.0, respectively. The enzyme was stable in the pH range 5.5-9.0 for 2 h over 80% and the temperature range 30-40 °C for 2 h more than 70%. The enzyme activity was inhibited by Ca2+, Fe2+, Cu2+, and Mg2+, especially Fe2+. The kinetic parameters of Km and Vmax were 1.38 mM and 24.64 µmol·mg-1·min-1 using pNPR as the substrate, respectively. When epimedin C was used as a nature substrate to determine the kinetic parameters of α-L-rhamnosidase, the values of Km and Vmax were 3.28 mM and 0.01 µmol·mg-1·min-1, respectively. The conditions of enzymatic hydrolysis were optimized through single factor experiments and response surface methodology. The icariin yield increased from 61% to over 83% after optimization. The enzymatic hydrolysis method could be used for the industrialized production of icariin. At the same time, this enzyme could also cleave the α-1,2 glycosidic linkage between glucoside and rhamnoside in naringin and neohesperidin, which could be applicable in other biotechnological processes.

HPLC-DAD Fingerprints Combined With Multivariate Analysis of Epimedii Folium From Major Producing Areas in Eastern Asia: Effect of Geographical Origin and Species.

The growth location and plant variety may influence the active components and biological activities of plants used in phytomedicine. In this study, nine sets of different Epimedii Folium, from different representative cultivation locations and Epimedium species, were collected for comparison, using HPLC-DAD combined with multivariate analysis. The objective was to investigate the influence of geographical origin and Epimedium species on the quality of Epimedii Folium, and provide applicable guidance for cultivation and quality control of Epimedii Folium. Several Epimedium spp. sets were used to establish the HPLC-DAD fingerprints and 91 peaks (compounds) were selected for the multivariate analysis. Major compounds were analyzed by HPLC-DAD combined with principal component analysis (PCA). HPLC quantitative analysis of known bioactive compounds was performed. Application of PCA to HPLC data showed that Epimedium samples sharing the same geographical origin or species clustered together, indicating that both species and geographical origin have impacts on the quality of Epimedii Folium. The major bioactive flavonoid compounds, epimedin C, icariin and baohuoside I, were identified and quantified. The concentration of bioactive compounds was significantly influenced both by species and geographical origin. E. sagittatum from Sichuan showed the highest content of bioactive compounds. The results showed that both Epimedium species and geographical origin have strong impact into quality of Epimedii Folium. HPLC data combined with multivariate analysis is a suitable approach to inform the selection of cultivation areas and choose Epimedium spp. most suitable for different geographical areas, resulting in improved quality of Epimedii Folium.

UPLC-MS/MS method for simultaneously determining nucleosides and methyl-nucleosides in liver mRNA of Epimedin C-induced liver injury mouse model.

BACKGROUND: Epimedin C, one of the main active ingredients of Epimedium, has been reported to have potential hepatotoxicity. However, the mechanism of Epimedin C-induced liver injury has not been studied. mRNA methylation, mainly including N6-methyladenosine and N5-methylcytidine, is implicated in the regulation of many biological processes and diseases. The study of quantifying mRNA methylation alterations in Epimedin C-induced liver injury mice may contribute to clarify the mechanism of its hepatotoxicity. Therefore, an analysis method needs to be established to determine nucleoside and methyl-nucleoside levels in liver mRNA. METHODS: An ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to simultaneously determine six nucleosides (adenosine, uridine, cytidine, guanosine, N6-methyladenosine and N5-methylcytidine) in liver mRNA. Besides, the Epimedin C-induced liver injury mouse model was studied by intragastrical administration Epimedin C at a daily dose of 10 or 40 mg/kg for 4 weeks. The nucleoside samples of the mice liver mRNA were prepared and separated on an UPLC column using 0.1% formic acid water and methanol after enzymatic digestion. Then the sample was detected by a Qtrap 6500 mass spectrometer. RESULTS: In this method, calibration curves of the six nucleosides showed good linearity over their concentration ranges. The linear ranges were 40-20,000 pg/mL for adenosine, cytidine, N6-methyladenosine and N5-methylcytidine, 0.2-100 ng/mL for guanosine, and 2-1000 ng/mL for uridine. Epimedin C-induced liver injury mouse model was successfully established,which could be proved by the elevation of serum aminotransferase levels, and the increased inflammatory cell infiltration as well as vacuolar degeneration in liver. The N6-methyladenosine and N5-methylcytidine levels, and the ratios of N6-methyladenosine to adenosine and N5-methylcytidine to cytidine of the mice liver mRNA were all significantly increased after Epimedin C treatment. CONCLUSION: The established method was successfully applied to the determination of six nucleosides levels in liver mRNA of the Epimedin C-induced liver injury mice model and the control group. The results indicated that mRNA methylation might be associated with Epimedin C-induced liver injury. This study will facilitate the mechanism research on the hepatotoxicity of Epimedin C.

Anti-Inflammatory and Chondroprotective Effects of Vanillic Acid and Epimedin C in Human Osteoarthritic Chondrocytes.

In osteoarthritis (OA), inhibition of excessively expressed pro-inflammatory cytokines in the OA joint and increasing the anabolism for cartilage regeneration are necessary. In this ex-vivo study, we used an inflammatory model of human OA chondrocytes microtissues, consisting of treatment with cytokines (interleukin 1ß (IL-1ß)/tumor necrosis factor α (TNF-α)) with or without supplementation of six herbal compounds with previously identified chondroprotective effect. The compounds were assessed for their capacity to modulate the key catabolic and anabolic factors using several molecular analyses. We selectively investigated the mechanism of action of the two most potent compounds Vanillic acid (VA) and Epimedin C (Epi C). After identification of the anti-inflammatory and anabolic properties of VA and Epi C, the Ingenuity Pathway Analysis showed that in both treatment groups, osteoarthritic signaling pathways were inhibited. In the treatment group with VA, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling was inhibited by attenuation of the nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα) phosphorylation. Epi C showed a significant anabolic effect by increasing the expression of collagenous and non-collagenous matrix proteins. In conclusion, VA, through inhibition of phosphorylation in NF-κB signaling pathway and Epi C, by increasing the expression of extracellular matrix components, showed significant anti-inflammatory and anabolic properties and might be potentially used in combination to treat or prevent joint OA.

Epimedin C modulates the balance between Th9 cells and Treg cells through negative regulation of noncanonical NF-κB pathway and MAPKs activation to inhibit airway inflammation in the ovalbumin-induced murine asthma model.

Allergic asthma is a common airway inflammatory disease and mainly caused by abnormal immune responses to allergens and viruses. The precise mechanisms of airway inflammation and airway hyper-responsiveness (AHR) are still not completely understood. CD4+ helper T cells (Th cells) serve as critical regulators of allergic immunity. The imbalance between T helper 9 (Th9) cells and forkhead box protein 3 (Foxp3)+ regulatory T (Treg) cells may contribute to airway inflammation in asthma. Epimedin C, a dominant compound isolated from Herba Epimedii, has shown anti-inflammatory effects and the immunoregulatory activity, such as increase of lymphocyte proliferation. However, the protective role of epimedin C in an experimental model of ovalbumin (OVA)-induced allergic airway inflammation and the underlying mechanism remain unknown. Female BALB/c mice were sensitized by intraperitoneal injection (i.p.) of OVA plus aluminum hydroxide (Alum) and subsequently challenged with an aerosol of 3% OVA in saline. Mice were treated with different concentrations of epimedin C (20 mg/kg/d, 40 mg/kg/d, 80 mg/kg/d) for 4 weeks. Experimental endpoints were evaluated via the analysis of AHR to acetyl-ß-methacholine (Mch), differential inflammatory cell counts, concentrations of cytokines interleukin-9 (IL-9), IL-4 and IL-10 in bronchoalveolar lavage fluid (BALF), serum OVA-specific IgE level, as well as airway inflammation, mucus secretion and collagen deposition in mice. Mechanistically, we investigated the percentages of Th9 cells and Treg cells, as well as mRNA levels of IL-9 and transcription factor Foxp3 in lungs. Furthermore, the proteins expression of nuclear factor-κB (NF-κB) family members p105/p50, RelA, p100/p52 and RelB, as well as mitogen-activated protein kinase (MAPK) family members extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPK was detected. Epimedin C dose-dependently attenuated AHR, airway inflammation, mucus hypersecretion and collagen deposition in OVA-induced murine asthma model. The expression levels of IL-9, IL-4 and OVA-specific IgE were significantly decreased while IL-10 was increased by epimedin C. We further confirmed that epimedin C decreased the percentage of lung Th9 cells with lower mRNA expression of IL-9 and increased the percentage of lung Treg cells with higher mRNA expression of Foxp3. In addition, epimedin C dose-dependently decreased the protein levels of p52, RelB, phosphorylation of ERK1/2 and p38 MAPK which are pivotal to the development of Th9 cells and Treg cells. Collectively, epimedin C could inhibit pathophysiological features of asthma by reconstruction of the balance between Th9 cells and Treg cells through regulation of the noncanonical NF-κB p52/RelB pathway and MAPKs activation. These findings suggest epimedin C as a potential remedy for inflammatory airway diseases.

Quality evaluation of different parts of multiple botanical origins of Epimedii Folium / 中草药

Objective: To compare the flavonoids contained in different parts of different botanical origins of Epimedii Folium, and provide a basis for the quality evaluation of Epimedii Folium and the reasonable selection of medicinal parts. Methods: The aerial parts of 13 batches of Epimedii Folium were collected and divided into three parts: leaf, petiole and stem. The HPLC fingerprint and content of five flavonoids, including epimedin A, epimedin B, epimedin C, icariin and baohuoside I, were analyzed. Then the analysis of variance and the similarity evaluation software of traditional Chinese medicine chromatographic fingerprint were used. Combined with cluster analysis (HCA), the content differences of flavonoids in leaf, petiole and stem of Epimedii Folium were evaluated. Results: The fingerprints showed that the chemical constituents in Epimedii Folium leaves were richer than stems and petioles, and the chemical constituents in petioles and stems were basically the same. The content of five components in leaves was significantly higher than that of petiole and stem, even up to 10 times. Cluster analysis also showed that the leaves were obviously distinct from the petiole and stem. Conclusion: The quality differences of Epimedii Folium leaves, petioles and stems were clarified, and this study can provide the scientific evidence for the selection of medicinal parts and quality control of Epimedii Folium.

Analysis changes of Epimedii Folium's flavonoids before and after processing based on UPLC-Q/TOF-MS / 中草药

Objective: Based on UPLC-Q/TOF-MS technology, the fingerprint of Epimediumkoreanum before and after processing was established to analyze the whole composition and find out the iconic chemical composition to clarify the change rule of flavonoids. Methods: The data of E.koreanum raw and processed products were collected in positive ion mode using UPLC-Q/TOF-MS technology, and orthogonal partial least least squares-discriminant analysis (OPLS-DA) method was used to explore the differences in chemical composition of E.koreanum before and after processing in nine different origins and batches. Results: Nine iconic chemical constituents from E.koreanum raw and processed products were found and identified, including 8-ethylene-kaempferol, icaritin, icariin I, icartin-3-O-glucoside, isoamyl alcohol sagittatoside B,1,3-prenyl epimedin C, 1,3-prenyl-sagittatoside B-7-O-glucuronic acid, 3-O-((4-acetoxy) rhamnose-2-O-(m-2acetoxy) glucose) icariin and its isomers. Conclusion: The structures of E.koreanum's flavonoids changed after the processing, the secondary glycosides of flavonoids increased, the polyglycosides decreased, and the flavonoids were generally converted to low glycoside components, which further clarified the change rule of E.koreanum's flavonoids after processing.

Purification process of epimedin and icariin from Epimedii Folium by macroporous resin / 中草药

Objective: To investigate the best technological conditions for the purification of epimedin and icariin from Epimedii Folium by macroporous resin, and preliminarily characterize the purification fraction of the best technological conditions. Methods: Five kinds of macroporous resins were screened by static adsorption experiment with the content of epimedin A1, epimedin A, epimedin B, epimedin C and icariin as indexes. The best purification conditions were optimized by the concentration of upper column solution, the maximum sample volume, the upper column flow rate, the volume of water washing, the concentration of removing impurity ethanol and elution ethanol, the volume of removing impurity ethanol and elution ethanol, the column diameter-height ratio through dynamic adsorption experiment. Finally, UPLC-Q-TOF/MS, HPLC and ultraviolet spectrophotometry were used to characterize the purification fraction of the best technological conditions. Results: The best macroporous resin was AB-8, column diameter-height ratio was 1:7, 6 BV of upper column solution (crude drug 0.5 g/mL) was used for dynamic adsorption at a flow rate of 6 BV/h, 5 BV of water and 5 BV of 20% ethanol were used for impurity removal, and 6 BV of 50% ethanol was used for elution. The flow rate of impurity removal and elution was 6 BV/h. After purification, the total flavonoids content was 63.29%, the total content of epimedin A1, A, B, C and icariin was 40.48%, the content of epimedin A1, epimedin A, epimedin B, epimedin C and icariin was 1.63%, 2.52%, 16.36%, 5.51% and 14.46%, respectively. Conclusion: The purification process of epimedin and icariin from Epimedii Folium by AB-8 macroporous resin is stable, reasonable and feasible. The chemical characterization indicated that the purification fraction was mainly flavonoids, mainly consisting of epimedin and icariin. The optimized purification process can be used for the purification and enrichment of such ingredients.

HPLC characteristic chromatograms and quantitative analysis of Kunxian Capsules / 中草药

Objective: In order to provide a scientific basis for the quality control of Kunxian Capsules (KC), HPLC characteristics chromatogram combined with multi-components determination was established. Methods: The analysis was performed on Agilent Zorbax SB-C18 column (250 mm × 4.6 mm, 5 μm), using acetonitrile and 0.1% phosphoric acid solution as the mobile phase at a flow rate of 0.8 mL/min for gradient elution, the column temperature was 33 ℃, and the detection wavelength was 270 nm. The fingerprints of 15 batches of KC were established and evaluated by the similarity evaluation system of TCM (2012A version), hierarchical cluster analysis and discriminant analysis of partial least squares. Furthermore, the content of hyperoside, epimedin A, epimedin B, epimedin C, icariin and baohuoside Ⅰ were determined. Results: The HPLC fingerprint with 21 common peaks of KC was established, and the similarities of samples were over 0.9. The linearity relationships separated with hyperoside, epimedin A, epimedin B, epimedin C, icariin and baohuoside Ⅰ were good, and the contents of the above-mentioned components in 15 batches of preparations were 2.817-7.527, 7.287-9.103, 8.730-18.675, 33.377-70.371, 35.297-50.291 and 4.059-9.079 mg/g, respectively. Conclusion: The combination methods of HPLC characteristic chromatograms and simultaneous determinations of multiple components are rapid, simple and reproducible, which can provide methodological reference for the quality control of KC.

Efficient bioconversion of epimedin C to icariin by a glycosidase from Aspergillus nidulans.

Herba Epimedii is a traditional Chinese herbal medicine that contains a mixture of bioactive flavonoid glycosides. Among them, icariin has the most outstanding bioactive functions, while epimedin C exhibits substantial toxicity. A recombinant α-L-rhamnosidase (synAnRhaE) from Aspergillus nidulans was expressed in Escherichia coli to promote the efficient bioconversion of epimedin C to icariin. A hydrolase activity of 574.5 U L-1 was acquired via optimized fed-batch fermentation in a 5-L bioreactor. The enzyme proved to be stable in an acidulous pH range below 55 °C with an optimal pH of 4.5 and optimal temperature of 55 °C. Epimedin C (1 g L-1) was 100% converted to icariin within 90 min using recombinant cells. The resting cells proved to be selective for epimedin C and 2″-O-rhamnosylicariside II in crude extracts of the epimedium plant. This work provides an original and efficient biocatalyst system that can be applied in industrialized production of icariin.

Epimedin C Protects H2O2-Induced Peroxidation Injury by Enhancing the Function of Endothelial Progenitor HUVEC Populations.

Endothelial cell injury and apoptosis induced by oxidative stress serve important roles in many vascular diseases. The repair of endothelial cell vascular injury relies on the function of local endothelial progenitor cells (EPCs). Our previous study indicated that epimedin C, a major flavonoid derived from Herba epimedii (yin yang huo), could promote vascularization by inducing endothelial-like differentiation of mesenchymal stem cells C3H/10T1/2 both in vivo and in vitro. In view of the significant cardiovascular protective effects of Herba epimedii, we detected a protective effect of epimedin C on hydrogen peroxide (H2O2)-induced peroxidation injury in human umbilical vein endothelial cells (HUVECs) and the role of EPC in this process. The results show that epimedin C increased the expression of the stem cell marker, CD34 and PROM1, and subsequently enhanced the expression and function of vascular endothelial growth factor and matrix metalloproteinase (MMP)-2 in local vascular endothelial cells. In conclusion, epimedin C protects H2O2-induced peroxidation injury by enhancing the function of endothelial progenitor HUVEC populations.

Integrated pathological cell fishing and network pharmacology approach to investigate main active components of Er-Xian decotion for treating osteoporosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Oxidative damage to osteoblasts was a key factor in the development of osteoporosis. Er-Xian Decotion (EXD) is widely used in China for the treatment of osteoporosis, which has a variety of antioxidant active ingredients. EXD may be an important source of protection against oxidative damage in osteoblasts, but the anti-osteoporotic active components of EXD is currently unclear. AIM OF THE STUDY: This work established an effective and reliable drug screening method to find main active ingredients in EXD (M-EXD) that can protect osteoblasts against oxidative stress and achieve anti-osteoporosis effects. MATERIALS AND METHODS: H2O2-induced osteoblast cell fishing with UHPLC-QTOF/MS was firstly used to discover the potential active components from EXD. Afterword, the EXD compound-osteoporosis target network was constructed using network pharmacology, thus potentially anti-osteoporosis ingredients were founded, and their combination were defined as the M-EXD. Finally, pharmacology effects of M-EXD was evaluated by ovariectomized rats, prednisolone induced-zebrafish and H2O2-induced osteoblasts. RESULTS: 40 candidate active ingredients in EXD were initially screened out via pathological cell fishing. According to network pharmacology result, M-EXD consisted of 13 ingredients since they had a close relationship with 65 osteoporosis-related targets. Pharmacological evaluation showed that M-EXD significantly ameliorated oxidative stress in H2O2-induced osteoblast model, evidently reversed the activity of ALP, ROS, GSH-px, NO and MDA compared with the model group. M-EXD showed better anti-oxidative activities than individual ingredients, presenting obvious synergetic effects. In osteoporosis rat and zebrafish models, M-EXD also demonstrated good anti-osteoporotic properties by mitigating the osteoporosis bone loss and increasing serum bone morphogenetic protein 2, and reversing osteocalcin expression in bone tissue. It significantly ameliorated oxidative stress in the in-vivo models. Moreover, M-EXD and EXD showed similar anti-osteoporotic and anti-oxidative properties, while the rest components of EXD had no satisfactory anti-osteoporotic efficacy. CONCLUSIONS: Our work successfully identified the main active components in EXD, which could represent the efficacy of EXD on treating osteoporosis, and meanwhile, it also provided an effective strategy to investigate active ingredients from natural medicines, which might be helpful for drug development and application.

Characterization of a α-l-rhamnosidase from Bacteroides thetaiotaomicron with high catalytic efficiency of epimedin C.

In this study, a α-l-rhamnosidase gene from Bacteroides thetaiotaomicron VPI-5482 was cloned and expressed in Escherichia coli. The specific activity of rhamnosidase was 0.57 U/mg in LB medium with 0.1 mM Isopropyl ß-d-Thiogalactoside (IPTG) induction at 28 °C for 8 h. The protein was purified by Ni-NTA affinity, which molecular weight approximately 83.3 kDa. The characterization of BtRha was determined. The optimal activity was at 55 °C and pH 6.5. The enzyme was stable in the pH range 5.0-8.0 for 4 h over 60%, and had a 1-h half-life at 50 °C. The Kcat and Km for p-nitrophenyl-α-l-rhamnopyranoside (pNPR) were 1743.29 s-1 and 2.87 mM, respectively. The α-l-rhamnosidase exhibited high selectivity to cleave the α-1,2 and α-1,6 glycosidic bond between rhamnoside and rhamnoside, rhamnoside and glycoside, respectively, which could hydrolyze rutin, hesperidin, epimedin C and 2″-O-rhamnosyl icariside II. Under the optimal conditions, BtRha transformed epimedin C (1 g/L) to icariin by 90.5% in 4 h. This study provides the first demonstration that the α-l-rhamnosidase could hydrolyze α-1,2 glycosidic bond between rhamnoside and rhamnoside.

Determination of ten flavones in Kunxian Capsules with quantitative analysis of multi-components by single marker / 中草药

Objective To establish quantitative analysis of multi-components by single marker (QAMS) method for simultaneous determining the content of ten flavonoids in Kunxian Capsules (KC), and evaluate the adaptation and application of QAMS method in the quality control of KC. Methods The relative factor (fs/i) of epimedin A, epimedin C, epimedin B, icariin, luteolin, quercetin, nobiletin, kaempferol and baohuoside I were established by HPLC method with hyperoside as internal standard, which were used to calculate the content of ten flavonoids in the samples of KC. Meanwhile, external standard method (ESM) was used to calculate the content of ten flavonoids. The difference between QAMS and ESM were analyzed to evaluate the accuracy of QAMS. T-test was used to compare the content of ten flavonoids in KC. Results The fs/i of epimedin A, epimedin C, epimedin B, icariin, luteolin, quercetin, nobiletin, kaempferol, and baohuoside I were 0.756 5, 2.199 4, 1.232 7, 1.008 5, 0.635 7, 0.576 0, 0.487 5, 0.545 6, 0.675 8. The content determination results of ten batches of KC samples were calculated by the method of QAMS and ESM, with no significant difference in RSD < 2.0%. Conclusion The fs/i established in the QAMS method with hyperoside as the internal reference substance is accurate and feasible. The QAMS method can be used for the quality evaluation of KC.