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Flavonoids such as baohuoside I and icaritin are the major active compounds in Epimedii Folium(EF)and possess excellent therapeutic effects on various diseases.Encouragingly,in 2022,icaritin soft capsules were approved to reach the market for the treatment of hepatocellular carcinoma(HCC)by National Medical Products Administration(NMPA)of China.Moreover,recent studies demonstrate that icaritin can serve as immune-modulating agent to exert anti-tumor effects.Nonetheless,both production effi-ciency and clinical applications of epimedium flavonoids have been restrained because of their low content,poor bioavailability,and unfavorable in vivo delivery efficiency.Recently,various strategies,including enzyme engineering and nanotechnology,have been developed to increase productivity and activity,improve delivery efficiency,and enhance therapeutic effects of epimedium flavonoids.In this review,the structure-activity relationship of epimedium flavonoids is described.Then,enzymatic en-gineering strategies for increasing the productivity of highly active baohuoside I and icaritin are dis-cussed.The nanomedicines for overcoming in vivo delivery barriers and improving therapeutic effects of various diseases are summarized.Finally,the challenges and an outlook on clinical translation of epi-medium flavonoids are proposed.
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Epimedii Folium is a traditional non-toxic Chinese herbal medicine. However, liver injury caused by Chinese herb preparations, including Epimedii Folium, is frequently reported over the years. Based on ancient and modern literature, this paper systematically summarized and analyzed the safe application of Epimedii Folium from the perspectives of varieties, processing methods, clinical adverse reactions, pharmacological effects and toxic mechanism. Combined with our team work, we build the comprehensive prevention and control system "human-drug-application", for the safe and rational application of Epimedii Folium. This study is expected to provide support for scientific evaluation and precise prevention and control of the safety risk of Epimedii Folium.
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There are many multi-original medicinal materials in Chinese Pharmacopoeia, and the mixed use of medicinal materials from different sources is common, which has certain influence on the stability of clinical medication. In this study, pyrosequencing technology was used to screen species-specific single nucleotide polymorphisms (SNP) from commonly used DNA barcode sequences, and a rapid and accurate molecular identification method for original species in mixed medicinal powder of Epimedii Folium was established. Multiple sequence alignment analysis showed that the 176th (C/T) mutation and the 196th (A/G) mutation of ITS, the 123rd (C/G) mutation of matK and the 892nd (A/C) mutation of rbcL could be used as the unique SNPs of E. sagittatum, E. koreanum, E. brevicornu and E. pubescens, respectively. In this study, the applicability of pyrosequencing and Sanger sequencing methods in the sequencing of mixture samples was investigated from the perspective of sensitivity and stability. Pyrosequencing method has higher detection sensitivity than Sanger sequencing method for low content samples in the mixed samples. Stability analysis showed that pyrosequencing technology could still obtain effective sequencing results for the amplified products of template DNA after 45 min of 95 ℃ high temperature water bath, while the critical point of Sanger sequencing method was 30 min. In this study, a new identification technology of Epimedii Folium mixed powder primordial species based on pyrosequencing and specific SNP was developed, which can quickly and accurately identify the mixed use of Epimedii Folium with high sensitivity and stability, and can also support the identification of different primordial species and mixed powder primordial herbs, which is conducive to ensuring the consistency and stability of clinical medication.
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Objective:To explore the effects of different processed products of Epimedii Folium on cytotoxicity and the material basis of toxicity. Methods:By using the SRB method to investigate the effects of different processed products of Epimedii Folium on the proliferation of HaCaT cells; and based on the grey correlation analysis method to establish the data spectrum effect relationship of HPLC fingerprint spectrum-toxicity so as to determine the toxic components and processing methods. Results:The value of cytotoxicity IC 50 of different processed products of Epimedii Folium is as follow: vinegar fried> oil fried > original > single fried > salt fried > wine fried. Among the 12 characteristic chromatographic peaks, Peak No.3 (magnoflorine, correlation value: 0.870) and Peak No.6 (epimedin C, correlation value: 0.851) are highly correlated with the toxicity value. Conclusions:Both vinegar fried and oil fried Epimedii Folium have the effect of reducing toxicity. Magnoflorine and epimedin C may be the main toxic components in Epimedii Folium. The study provides scientific basis for the research on the process optimization of Epimedii Folium concocting to reduce toxicity.
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Epimedii folium is a commonly used Traditional Chinese Medicine (TCM) for warming the kidney and strengthening the yang qi. It has androgen-estrogen-like effect. It can not only directly act on sexual organs to regulate hormone levels, but also exert sex-hormone-like effect through hypothalamus-pituitary-gonadal axis. Its regulation of hormone levels is similar to that of plant hormones. At present, Epimedii folium is commonly used with other TCMs to treat diseases caused by sex-hormone deficiency, such as male spermatopenia, asthenospermia, benign prostatic hyperplasia, functional erectile dysfunction, female premature ovarian failure, perimenopausal syndrome, dysfunctional infertility during ovulation, hyperandrogenemia of PCOS patients, etc.
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Liver cancer is a worldwide malignant tumor with an increasing incidence by years. At present， it is facing the predicament of poor prognosis and lack of effective therapeutic drugs. Epimedii Folium is a well-known traditional Chinese medicine with a long history， and exiting clinical and pharmacological studies show that it can be used in clinical treatment of liver cancer. According to reports， Epimedii Folium polysaccharides （EPS）， C-8-isopentenyl substituted flavonoids and their glycosides （icaritin， icariin， baohuoside Ⅰ， epimedin C） have good anti-liver cancer activity. They are the main active ingredients of Epimedii Folium against liver cancer. The data which comes from in vitro and in vivo studies suggests flavonoids in Epimedii Folium demonstrate anti-liver cancer activity through various mechanisms， including inhibiting hepatoma cells proliferation， promoting hepatoma cells apoptosis， improving tumor immunosuppression microenvironment， inhibiting hepatoma cells immune escape， invasion and migration， reversing hepatoma cells resistance， suppressing hepatocellular carcinoma initiation cells and regulating the immunity of the body. While EPS play an anti-hepatocellular carcinoma role mainly through the regulation of immunity. Epimedii Folium exerts good anti-liver cancer effects with multiple components， multiple targets， and multiple pathways， which makes it a valuable anti-liver cancer drug. However， the comprehensive analysis of related aspects is still lacking. Therefore， this study briefly reviewed the anti-hepatocellular carcinoma active ingredients of Epimedii Folium and their mechanisms. In addition， in the process of literature review， it was found that the anti-liver cancer studies of Epimedii Folium mainly focused on a few components and the studies elucidating the active constituents and mechanism of Epimedii Folium against liver cancer on the whole level were insufficient. Based on these questions， the study also proposed corresponding suggestion to provide reference for the further study of substance basis， clinical application and rational development of Epimedii Folium against liver cancer.
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ObjectiveTo explore the effective components and mechanism of Epimedii Folium in the treatment of oligoasthenotspermia by using network pharmacology and molecular docking technique. MethodThe main active components and corresponding target genes of Epimedii Folium were screened out from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）. Target genes of oligospermia were obtained by GeneCards and Online Mendelian Inheritance in Man （OMIM） database. Uniprot was used to correct all genes. The drug-active component-key target regulatory network was constructed by Cytoscape3.9.0， and the key active components were screened out according to the degree value. The active components and common targets of the disease were uploaded to STRING 11.5 database to construct the Epimedii Folium and oligoasthenotspermia target protein-protein interaction （PPI） network， and the key protein targets were screened out according to the degree value. The key targets of gene ontology （GO） functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis were performed using DAVID database. Protein Data Bank （PDB） and TCMSP were used to obtain the molecular structure of target proteins and active components. AutoDock Vina 1.1.2 was used to perform molecular docking of the active components and the core protein targets. Finally， icariin， the active component of Epimedii Folium， was used to intervene in the rat model of oligoasthenotspermia to verify the effect of icariin on the expression level of protein targets. ResultTwenty-three active components from Epimedii Folium were screened out， and 50 common targets and 6 core targets of oligoasthenotspermia and Epimedii Folium were obtained， including tumor protein p53 （TP53）， epidermal growth factor receptor （EGFR）， prostaglandin-endoperoxide synthase 2 （PTGS2）， cysteine aspartate-specific protease （Caspase）-3， erb-b2 receptor tyrosine kinase 2 （ERBB2）， and caspase-9. Through GO enrichment and KEGG pathway enrichment analysis， the active components of Epimedii Folium were mainly involved in the P53 signaling pathway， the pathways in cancer， cell proliferation， and apoptosis， etc. Molecular docking results indicated that icariin， quercetin， and 8-isopentenol had strong binding ability to target protein. The results of icariin intervention experiment showed that as compared with the control group， the expression of target proteins in testis of rats with oligoasthenotspermia was significantly down-regulated. As compared with the model group， icariin significantly up-regulated the expression of target protein in testis of rats with oligoasthenotspermia （P<0.05）. ConclusionEpimedii Folium treats oligoasthenotspermia through regulating the P53 signaling pathway， the pathways in cancer， cell proliferation， and apoptosis by icariin， quercetin， and 8-isopentenol.
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ObjectiveTo explore the effective components and mechanism of Epimedii Folium in the treatment of oligoasthenotspermia by using network pharmacology and molecular docking technique. MethodThe main active components and corresponding target genes of Epimedii Folium were screened out from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP）. Target genes of oligospermia were obtained by GeneCards and Online Mendelian Inheritance in Man （OMIM） database. Uniprot was used to correct all genes. The drug-active component-key target regulatory network was constructed by Cytoscape3.9.0， and the key active components were screened out according to the degree value. The active components and common targets of the disease were uploaded to STRING 11.5 database to construct the Epimedii Folium and oligoasthenotspermia target protein-protein interaction （PPI） network， and the key protein targets were screened out according to the degree value. The key targets of gene ontology （GO） functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment analysis were performed using DAVID database. Protein Data Bank （PDB） and TCMSP were used to obtain the molecular structure of target proteins and active components. AutoDock Vina 1.1.2 was used to perform molecular docking of the active components and the core protein targets. Finally， icariin， the active component of Epimedii Folium， was used to intervene in the rat model of oligoasthenotspermia to verify the effect of icariin on the expression level of protein targets. ResultTwenty-three active components from Epimedii Folium were screened out， and 50 common targets and 6 core targets of oligoasthenotspermia and Epimedii Folium were obtained， including tumor protein p53 （TP53）， epidermal growth factor receptor （EGFR）， prostaglandin-endoperoxide synthase 2 （PTGS2）， cysteine aspartate-specific protease （Caspase）-3， erb-b2 receptor tyrosine kinase 2 （ERBB2）， and caspase-9. Through GO enrichment and KEGG pathway enrichment analysis， the active components of Epimedii Folium were mainly involved in the P53 signaling pathway， the pathways in cancer， cell proliferation， and apoptosis， etc. Molecular docking results indicated that icariin， quercetin， and 8-isopentenol had strong binding ability to target protein. The results of icariin intervention experiment showed that as compared with the control group， the expression of target proteins in testis of rats with oligoasthenotspermia was significantly down-regulated. As compared with the model group， icariin significantly up-regulated the expression of target protein in testis of rats with oligoasthenotspermia （P<0.05）. ConclusionEpimedii Folium treats oligoasthenotspermia through regulating the P53 signaling pathway， the pathways in cancer， cell proliferation， and apoptosis by icariin， quercetin， and 8-isopentenol.
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Flavonoid glycosides are the main active constituents of Epimedii Folium and its related plants. They can be divided into polyglycosides and low glycosides according to the number of glycosyl group. The polyglycosides of Epimedii Folium can be transformed into low glycosides after biotransformation ；pharmacological activities of low glycosides in anti-tumor ，tonifying kidney yang and anti-osteoporosis are stronger than those of polyglycosides. In this paper ， the research progress about biotransformation technology of flavonoid glycosides of Epimedii Folium was reviewed. It was found that the main biotransformation pathway of flavonoid glycosides of Epimedii Folium was to obtain low glycosides by removing glycosyl group ； related methods were mainly enzymatic hydrolysis and microbial transformation ，and also included plant cell transformation ，acid hydrolysis method and synthesis method.
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Objective:This study by using the method of network pharmacology to screen the active constituent and related targets of Epimedii Folium aims to explore the mechanisms of the reinforcing effect of Epimedii Folium on kidney in the treatment of PCOS. Methods:By retrieving data from TCMSP datebase, screened out the active constituent of Epimedii Folium and the information of the targets corresponding to each active constituent; by using the gene database of NCBI, translated the information of the targets into gene names; by retrieving data from GeneCards datebase, extracted the genes related to PCOS; related targets of Epimedii Folium in the treatment of PCOS were obtained by Venn tool; by using Cytoscape 3.7.2 software, constructed a network diagram of Epimedii Folium-active constituents-targets-PCOS; by using STRING database, constructed the protein interaction network; then carried out GO enrichment analysis of related targets by Geneontology database and carried out pathway enrichment analysis of related targets by KEGG database. Results:There were 23 active constituents of Epimedii Folium and 132 related targets treating PCOS. The Epimedii Folium could play the reinforcing effect on kidney mainly by regulating the biological processes like steroid hormone receptor activity, as well as KEGG pathways such as Estrogen signaling pathway, GnRH signaling pathway, GnRH secretion, HIF-1 signaling pathway and VEGF signaling pathway in treating PCOS. Conclusion:From the perspective of network pharmacology, this study preliminarily analyzed the related targets and pathways of reinforcing effect on kidney of Epimedii folium in the treatment of PCOS, providing reference for further experiments and application inclinics.
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Objective:To explore the mechanism of the prescription consisting Aconiti Lateralis Radix Praeparata and Epimedii Folium in the treatment of chronic heart failure （CHF） based on network pharmacology，followed by verification in H9c2 myocardial cells with hypoxia-reoxygenation injury <italic>in vitro</italic> and in zebrafish with vascular endothelial growth factor receptor （VEGFR） tyrosine kinase inhibitor Ⅱ （VRI） -induced vascular insufficiency. Method:The active ingredients in Aconiti Lateralis Radix Praeparata and Epimedii Folium were searched from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP），the corresponding target genes from the Universal Protein Resource （UniProt）， and the CHF-related targets from Online Mendelian Inheritance in Man （OMIM） and GeneCards. Both the active ingredient-potential target network and the active ingredient-CHF-related target network were generated using Cytoscape 3.6.1， followed by the protein-protein interaction （PPI） network construction and Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genome （KEGG） enrichment analysis based on MetaScape. H9c2 myocardial cells exposed to hypoxia-reoxygenation were selected for determining the proliferation-promoting effect by methyl thiazolyl tetrazolium （MTT） assay. The protein expression of B-cell lymphoma-2（Bcl-2）， Bcl-2-associated X protein（Bax），cysteinyl aspartate-specific protease-3（Caspase-3）， protein kinase B（PKB/Akt），phosphorylated protein kinase B（p-Akt），phosphorylated extracellular signal-regulated kinases 1/2 （p-ERK1/2），extracellular signal-regulated kinase 1/2 （ERK1/2）， and poly adenosine diphosphate ribose polymerase（PARP）was detected by Western blotting. The efficacy of the prescription in promoting angiogenesis was verified in a zebrafish model of VRI-induced vascular injury. Result:There were 28 active ingredients for the prescription， 209 corresponding targets， 1 296 CHF-related targets， and 94 common gene targets shared by the prescription and CHF. PPI network clustering suggested that Aconiti Lateralis Radix Praeparata and Epimedii Folium alleviated CHF by interfering with cell differentiation and metabolism and angiogenesis. GO analysis revealed that CHF relief was achieved via the intervention in such biological processes as cell migration，vascular development， and angiogenesis. Pharmacodynamic experiments verified that Epimedii Folium （10 mg·L<sup>-1</sup>） alone and the prescription （10 mg·L<sup>-1</sup>）both enhanced the proliferation of H9c2 myocardial cells under the hypoxia-reoxygenation condition （<italic>P</italic><0.05），while the latter also increased the expression of Bcl-2，Bcl-2/Bax， and PARP （<italic>P</italic><0.05） and reduced the expression of Caspase-3， Akt， and ERK （<italic>P</italic><0.05）. The prescription at the concentrations of 0.3 and 0.1 g·L<sup>-1</sup> promoted angiogenesis （<italic>P</italic><0.05）. Conclusion:Aconiti Lateralis Radix Praeparata and Epimedii Folium exert the therapeutic effect against CHF via multiple ingredients，multiple targets， and multiple channels. Such combination promotes the proliferation of H9c2 myocardial cells under hypoxic condition and protects zebrafish from vascular injury by up-regulating the expression of Bcl-2 and PARP，increasing Bcl-2/Bax ratio，and down-regulating the expression of Caspase-3，Akt， and ERK.
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The quality control of Epimedii Folium, composed of diverse constituents, is single at present. In view of this, an eva-luation method of 13 chemical constituents based on quantitative analysis of multi-components by single marker(QAMS) was established to further explore the composition differences of raw products and alcohol extracts in different batches and the influence of alcohol extraction on the composition, so as to provide a reference for improving the quality evaluation and control of Epimedii Folium. The fingerprints of different batches of Epimedii Folium were constructed by ultra-high performance liquid chromatography(UPLC) to evaluate the inter-batch consistency. The changes of the flavonoids in Epimedii Folium during alcohol extraction were analyzed based on determined levels and heat map, and the reasons for the changes were preliminarily discussed. With icariin, the quality control component recorded in the Chinese Pharmacopoeia, as the internal reference, the stability of the relative correction factors of chemical components under different conditions was investigated to obtain the relative correction factors. Then the determination results of QAMS and the external standard method were compared to verify the accuracy of QAMS. The results revealed that all batches of Epimedii Folium met the requirements specified in the Chinese Pharmacopoeia, and the fingerprints of Epimedii Folium from the same place of origin exhibited a high similarity. Raw products and alcohol extracts of Epimedii Folium could be clearly distinguished by prenylated flavonoids, which are potential biomarkers for quality control. Additionally, the glycoside hydrolysis in the alcohol extraction was preliminarily explored. The QAMS method has good accuracy, durability, and repeatability in determining 13 chemical components in Epimedii Folium under different experimental conditions. No significant difference in the results obtained by the two methods was observed. This study can provide a reference for comprehensive, rapid and reasonable quality evaluation of Epimedii Folium.
Subject(s)
Biomarkers , Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Plant LeavesABSTRACT
Xianling Gubao is a common and effective medicine in the treatment of orthopedic diseases. In recent years, it has been reported to be associated with liver injury. However, through the analysis of the adverse drug reaction reports and key hospital cases, we found that there is considerable incomplete information in the reports of Xianling Gubao-related liver injury cases retrieved from the literature. Thus, it is difficult to accurately judge causality between the drug and liver injury. Six cases of liver injury related to Xianling Gubao were identified in key hospitals, two of which achieved the clinical diagnosis according to the assessment of the integrated evidence chain method. We further analyzed the public health data of all residents in Yinzhou. The gross incidence rate of Xianling Gubao-related liver injury was 0.034%, which corresponds to a level of rare incidence. This revealed that Xianling Gubao-related liver injury has significant divergence in individuals and an idiosyncratic nature. The gross incidence of liver injury related to Xianling Gubao was lower than that of other medicines for the treatment of orthopedic diseases. Based on the idiosyncratic drug-induced liver injury model mediated by immune stress, it was found that Epimedii Folium and Psoraleae Fructus were the major components that lead to liver injury, and the liver injury caused by a full prescription was less serious than that encountered with only Epimedii Folium and Psoraleae Fructus. This suggests that the other 4 herbs (Dipsaci Radix, Anemarrhenae Rhizoma, Rehmanniae Radix,Salviae Miltiorrhizae Radix et Rhizoma) can prevent/alleviate the liver injury. Through disassembled prescription analysis, we found that the attenuation efficacy of Salviae Miltiorrhizae Radix et Rhizoma was the most significant. In conclusion, Xianling Gubao may cause idiosyncratic liver injury in a tiny minority of susceptible individuals, but the incidence risk is lower than that of other commonly used drugs for orthopedic disease. Xianling Gubao should be discreetly applied to patients with immune stress. The major components that induced liver injury in Xianling Gubao were Epimedii Folium and Psoraleae Fructus, and Salviae Miltiorrhizae Radix et Rhizoma appears to attenuate this toxicity. This study provides a reference for the rational clinical medication with Xianling Gubao.
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Alzheimer's disease(AD) patients in China have been surging, and the resultant medical burden and care demand have a huge impact on the development of individuals, families, and the society. The active component compound of Epimedii Folium, Astragali Radix, and Puerariae Lobatae Radix(YHG) can regulate the expression of iron metabolism-related proteins to inhibit brain iron overload and relieve hypofunction of central nervous system in AD patients. Hepcidin is an important target regulating iron metabolism. This study investigated the effect of YHG on the expression of a disintegrin and metalloprotease-17(ADAM17), a key enzyme in the hydrolysis of β amyloid precursor protein(APP) in HT22 cells, by mediating hepcidin. To be specific, HT22 cells were cultured in vitro, followed by liposome-mediated siRNA transfection to silence the expression of hepcidin. Real-time PCR and Western blot were performed to examine the silencing result and the effect of YHG on hepcidin in AD cell model. HT22 cells were randomized into 7 groups: control group, Aβ25-35 induction(Aβ) group, hepcidin-siRNA(siRNA) group, Aβ25-35 + hepcidin-siRNA(Aβ + siRNA) group, Aβ25-35+YHG(Aβ+YHG) group, hepcidin-siRNA+YHG(siRNA+YHG) group, Aβ25-35+hepcidin-siRNA+YHG(Aβ+siRNA+YHG) group. The expression of ADAM17 mRNA in cells was detected by real-time PCR, and the expression of ADAM17 protein by immunofluorescence and Western blot. Immunofluorescence showed that the ADAM17 protein expression was lower in the Aβ group, siRNA group, and Aβ+siRNA group than in the control group(P<0.05) and the expression was lower in the Aβ+siRNA group(P<0.05) and higher in the Aβ+YHG group(P<0.05) than in the Aβ group. Moreover, the ADAM17 protein expression was lower in the Aβ+siRNA group(P<0.05) and higher in the siRNA+YHG group(P< 0.05) than in the siRNA group. The expression was higher in the Aβ+siRNA+YHG group than in the Aβ+siRNA group(P<0.05). The results of Western blot and real-time PCR were consistent with those of immunofluorescence. The experiment showed that YHG induced hepcidin to up-regulate the expression of ADAM17 in AD cell model and promote the activation of non-starch metabolic pathways, which might be the internal mechanism of YHG in preventing and treating AD.
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Humans , ADAM17 Protein , Alzheimer Disease/genetics , Amyloid beta-Peptides , Drugs, Chinese Herbal/pharmacology , Hepcidins/genetics , PuerariaABSTRACT
To probe the potential hepatotoxic components of Epimedii Folium and investigate its mechanism based on network toxicology and cell experimental validation. According to the previous results of component measurement and cytotoxicity evaluation, 11 active compounds related to hepatotoxicity in Epimedii Folium were chosen as research object in this study. Through SwissTargetPrediction database and GeneCards database, the potentially hepatotoxic targets of Epimedii Folium were obtained. Subsequently, the protein-target interaction network and active compounds-hepatotoxic targets network were established to analyze the core targets and screen the key hepatotoxic compounds in Epimedii Folium. Meanwhile, the signaling pathways and molecular mechanisms were inferred with GO functional enrichment analysis and KEGG pathway enrichment analysis on the core targets. At last, the effect of icaritin as the chief hepatotoxic compound on the indexes related to hepatotoxicity in HL-7702 cells and HepG2 cells was investigated to validate the hepatotoxicity mechanism of Epimedii Folium. Through the network toxicology analysis, 190 action targets and 991 hepatotoxic targets were collected, then 64 potentially hepatotoxic targets of Epimedii Folium including AKT1, EGFR, MAPK3, TNF and so on were obtained, and icaritin was screened as the key hepatotoxic compound. GO functional enrichment analysis indicated 160 biological process terms such as protein phosphorylation and negative regulation of apoptotic process, 41 molecular function terms such as protein binding and ATP binding, and 32 cellular component terms such as cytosol and cell surface. KEGG pathway enrichment analysis inferred 75 signaling pathways involving PI3 K-Akt and HIF-1. After comprehensive analysis, it was inferred that the hepatotoxicity mechanism of Epimedii Folium was related with regulating oxidative stress and apoptosis. The results of cell biology experiments showed that icaritin could significantly increase the level of aspartate aminotransferase and lactate dehydrogenase, reduce the level of glutathione, improve the quality of reactive oxygen species and reduce mitochondrial membrane potential, indicating that it could cause hepatotoxicity by destroying cell membrane structure, inhibiting antioxidant enzyme activity, activating oxidative stress and inducing apoptosis. These results proved the reliability of results of network pharmacology. This study preliminarily clarified the material base and the mechanism of potential hepatotoxicity of Epimedii Folium, which provided important information for further research and safe application.
Subject(s)
Drugs, Chinese Herbal/toxicity , Plant Leaves , Protein Interaction Maps , Reproducibility of ResultsABSTRACT
Objective:To elucidate the potential molecular markers and drug-compound-target mechanism of Epimedii Folium intervention on breast cancer stem cells（BCSCs） through chip analysis combined with network pharmacology and experimental validation. Method:Relevant drug information was retrieved in Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） to obtain the active components and potential targets of Epimedii Folium. "Breast Cancer Stem Cells" were searched in Gene Expression Omnibus（GEO）database，and GSE98239 chip data were obtained through analysis and screening. Then GEO2R online analysis tool was used to obtain the differential genes to draw differential gene heat map and volcano map. The differential gene network map of Epimedii Folium intervention for breast cancer stem cells was constructed by Cytoscape 3.8.0，and Gene Ontology（GO） and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis of drug and disease genes were performed. Human breast cancer MDA-MB-231 cells were divided into 20%，40%，60% Epimedii Folium drug-containing serum group and control group. Cell counting kit-8（CCK-8），and Western blot were used to detect the effect of Epimedii Folium drug-containing serum intervention on cell activity and target protein expression in breast cancer cells. Result:Twenty-three active components including flavones，sterols，alkaloids and sesquiterpenoids were obtained from Epimedii Folium. It was found that Epimedii Folium interacted with B-cell lymphoma-2-like protein 1（BCL2L1），matrix metallopeptidase 2（MMP2），prostaglandin-endoperoxide synthase 2（PTGS2），vascular endothelial growth factor A（VEGFA），transforming growth factor beta receptor 1（TGFBR1） and other pivotal genes in breast cancer stem cells，participated in the induction of new angiogenesis and cell migration，enabled the continuous self-renewal of BCSCs，decreased apoptosis and cell migration，thus promoting the recurrence and metastasis of breast cancer. KEGG results showed that Epimedii Folium intervened in multiple differential expressed genes（DEGs）of transforming growth factor-<italic>β</italic>（TGF-<italic>β</italic>），vascular endothelial growth factor（VEGF），phosphoinositide 3kinase/protein kenase B（PI3K/Akt），mitogen-activated protein kinese（MAPK）and mammalian target of rapamycin（mTOR）subpathways in cancer signaling pathways to exert its efficacy in intervening breast cancer stem cells. Experiments showed that the survival rate of breast cancer cells was significantly reduced and the expression levels of TGFBR1 and Smad2 in breast cancer cells significantly decreased after the intervention of Epimedii Folium drug-containing serum（<italic>P</italic><0.01）. Conclusion:Several components in different concentrations of drug-containing serum of Epimedii Folium can synergistically act on target differentially expressed genes of breast cancer stem cells，and inhibit the proliferation of breast cancer cells by down-regulating the expression levels of TGFBR1，a key molecule in the TGF-<italic>β</italic> pathway，and Smad2，a downstream signal.
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Objective:To observe the effect of total flavonoids from Epimedii Folium （TEF） on the angiogenesis of ischemic myocardium in rats after acute myocardial infarction （AMI） and discuss its molecular biological mechanism of attenuating myocardial ischemia and improving cardiac function. Method:AMI in rats was induced through the ligation of left anterior descending coronary artery. All male SD rats were randomized into sham-operated group， model group， diltiazem group （10 mg·kg<sup>-1</sup>·d<sup>-1</sup>）， and TEF low-dose and high-dose groups （100 and 200 mg·kg<sup>-1</sup>·d<sup>-1</sup>）， with 8 rats in each group. After modeling， rats in the diltiazem group and TEF groups were given corresponding doses of diltiazem and TEF， respectively， and those in the model group and sham-operated group received normal saline of equivalent volume， once a day for 7 days. After the administration， VisualSonics Vevo2100 imaging system was used to detect the cardiac structure and function and hematoxylin-eosin （HE） staining to observe the histomorphological changes in myocardial ischemic area. Immunohistochemistry was employed to analyze the expression of CD31 and <italic>α</italic>-smooth muscle actin （<italic>α</italic>-SMA） in ischemic myocardium and Western blot to detect the expression of vascular endothelial growth factor-receptor 2 （VEGF-R2） and phosphorylation of protein kinase B （Akt） in ischemic myocardium. Real-time PCR was applied to quantify the mRNA levels of vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF）. Result:Compared with the sham-operated group， the model group demonstrated significant increase in left ventricular systolic diameter （LVIDs）， left ventricular internal diameter at end-diastole （LVIDd）， left ventricular end-systolic volume （LVEVs）， and left ventricular end-diastolic volume （LVEVd）， significant decrease in End-systolic thickness of left ventricular anterior wall （LVAWs）， end-diastolic thickness of left ventricle anterior wall （LVAWd）， end systolic thickness of left ventricular posterior wall （LVPWs）， stroke volume （SV）， ejection fraction （EF）， fractional shortening （FS）， and cardiac output （CO）， obvious pathological changes in the ischemic myocardium， and plummet of the expression of CD31 and <italic>α</italic>-SMA （<italic>P</italic><0.01）， Akt phosphorylation level， protein level of VEGF-R2， and mRNA levels of VEGF and bFGF （<italic>P</italic><0.05， <italic>P</italic><0.01）. High-dose TEF significantly alleviated the pathological changes of ischemic myocardium as compared with the model group. Moreover， TEF high-dose group showed significantly lower levels of LVIDs， LVIDd， LVEVs， and LVEVd， significantly higher levels of LVAWs， LVAWd， LVPWs， SV， EF， FS， and CO， higher expression of CD31 and <italic>α</italic>-SMA （<italic>P</italic><0.05， <italic>P</italic><0.01）， and higher levels of VEGF-R2 protein， phosphorylated Akt， and VEGF and bFGF mRNA than the model group （<italic>P</italic><0.05， <italic>P</italic><0.01）. Conclusion:TEF can effectively improve myocardial perfusion in peri-myocardial infarction area and attenuate ventricular remodeling and heart failure after AMI by up-regulating the expression of bFGF， VEGF， and VEGF-R2 in ischemic myocardium following AMI and activating phosphatidylinositol 3-kinases （PI3K）/Akt/VEGF signaling transduction pathway which can promote angiogenesis in ischemic myocardium.
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Objective:To study the material basis and potential molecular mechanism of Epimedii Folium against osteoporosis. Method:The chemical components in 14 batches of Epimedii Folium were analyzed by ultra-performance liquid chromatography-quadrupole-time of flight-tandem mass spectrometry （UPLC-Q-TOF-MS/MS）. With the activity of alkaline phosphatase （ALP） as the pharmacodynamic index，the partial least squares regression analysis （PLSR） was conducted to establish a model uncovering the spectrum-effect relationship between UPLC-Q-TOF-MS/MS spectral peak and ALP activity and screen the active components against osteoporosis. Online databases such as the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP），Comparative Toxicogenomics Database （CTD），Database for Annotation，Visualization，and Integrated Discovery （DAVID） and Cytoscape 3.6.1 were employed to predict the possible mechanism of action of Epimedii Folium against osteoporosis. Result:A total of 61 peaks and 56 compounds were identified by UPLC-Q-TOF-MS/MS. PLSR showed that icariin，baohuoside Ⅰ，epimedin A，sagittatoside A，and baohuoside Ⅱ might be the active components for Epimedii Folium to inhibit osteoporosis considering their strong correlation with ALP activity. As revealed by the network pharmacological analysis of the five components mentioned above，Epimedii Folium<italic> </italic>mainly regulated seven targets such as tumor necrosis factor （TNF），androgen receptor （AR），and estrogen receptor 1 （ESR1） and eight key pathways like endocrine and other factor-regulated calcium reabsorption，vascular endothelial growth factor （VEGF） signaling pathway，and transient receptor potential （TRP） channels to exert its anti-osteoporosis effect. Conclusion:The exploration of material basis and potential molecular mechanism of Epimedii Folium against osteoporosis based on UPLC-Q-TOF-MS/MS，spectrum-effect relationship，and network pharmacology has provided an experimental basis for the scientific explanation and clinical application of Epimedii Folium in treating osteoporosis.
ABSTRACT
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.
ABSTRACT
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.