Clinical Characteristics and Potential Risk Factors Analysis of Liver Injury Related to Epimedii Folium Preparation / 中国实验方剂学杂志

ObjectiveThis paper aims to analyze the clinical characteristics and medication rationality of liver injury related to Epimedii Folium preparation （EP） and explore the possible risk factors of liver injury， so as to provide a reference for the safe clinical application of Epimedii Folium （EF）. MethodA retrospective analysis was conducted on liver injury cases related to EP from 2012 to 2016. ResultThe number of reported liver injury cases and the proportion of severe cases related to the use of EP show an increasing trend， indicating the objective existence of liver injury caused by EP. There are more cases of liver injury related to EP in women than in men， with an onset age range of 15-91 years old and a median onset age of 60 years old （median onset ages for men and women are 59 and 60 years old， respectively）. The time span from taking EP alone to the occurrence of liver injury is 1-386 days， with a median of 38 days. The time span from taking both EP and Western medicine to the occurrence of liver injury is 1-794 days， with a median of 34 days. EF-related liver injury preparations are mostly composed of traditional Chinese medicines that promote immunity and tonify the liver and kidney， indicating that immune stress in the body may be the mechanism of liver injury caused by the use of EP alone or in combination. There is no increasing trend of toxicity with time or dose in the liver injury caused by EP. By further exploring its risk factors， it is found that patients have unreasonable medication methods such as excessive dosage， repeated use， and multi-drug combination， which may also be one of the important risk factors for EF-related liver injury. ConclusionEP has a certain risk of liver injury and should be emphasized in clinical diagnosis and treatment. Immune stress may be the mechanism of liver injury caused by EP， and in clinical use， it is necessary to be vigilant about the risk of liver injury caused by unreasonable use and combined use with Western medicine.

Development of an in vitro screening method for idiosyncratic hepatotoxic components in traditional Chinese medicine: a case study with Epimedii Folium and Psoraleae Fructus / 药学学报

Idiosyncratic drug-induced liver injury (IDILI) has long posed a challenging and pivotal concern in pharmaceutical research. The complex composition of traditional Chinese medicine (TCM) has introduced a bottleneck in current research, hindering the elucidation of the component basis associated with IDILI in TCM. Using Epimedii Folium (EF) and Psoraleae Fructus (PF) as illustrative examples, this study endeavors to establish an in vitro evaluation model, providing a high-throughput and preliminary assessment method for screening components related to TCM-induced IDILI. A TNF-α-mediated HepG2 susceptible model was first established in this study, with the focus on the index components present in EF and PF. The release of lactate dehydrogenase (LDH) in the cell supernatant served as the detection index. A concentration-toxicity response curve was constructed, and the hepatotoxic components of EF and PF were identified utilizing the synergistic toxicity index. The LDH results unveiled the hepatotoxic effects of bavachin, backuchiol, isobavachin, neobavaisoflavone, psoralidin, isobavachalcone, icarisid I, and icarisid II on both normal and susceptible cells, categorizing these 8 components as both direct hepatotoxicity components and idiosyncratic hepatotoxicity components. Bavachin and neobavaisoflavone exhibited no hepatotoxicity on normal cells but demonstrated significant effects on susceptible cells, designating them as potential idiosyncratic susceptible hepatotoxicity components. The study further delineated that 10 EF components and 3 PF components were direct immune-promoting hepatotoxicity components. Additionally, 14 idiosyncratic immune-promoting hepatotoxicity components were identified, encompassing 10 EF components and 4 PF components, with neobavaisoflavone, bavachinin, and isobavachin being potential idiosyncratic susceptible immune-promoting hepatotoxicity components. Synergistic toxicity index results indicated that 13 idiosyncratic immune-promoting hepatotoxicity components (except anhydroicaritin) combined with bavachin demonstrated synergistic hepatotoxicity on susceptible cells. Notably, 3 idiosyncratic susceptible immune-promoting hepatotoxicity components combined with bavachin exhibited synergistic hepatotoxicity, with neobavaisoflavone displaying the highest synergistic toxicity index and bavachinin the lowest. In summary, this methodology successfully screens hepatotoxic and immune-promoting hepatotoxic components in EF and PF, distinguishing the types of components inducing hepatotoxicity, evaluating the hepatotoxicity degree of each component, and elucidating the synergistic relationships among them. Importantly, these findings align with the characteristics of IDILI. The method provides an effective model tool for the fundamental research of TCM-related IDILI components.

Effect and Mechanism of Action of Epimedii Folium Polysaccharides on Mice with Exercise-induced Fatigue Based on p38 MAPK/NF-κB Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo study the effects of Epimedii Folium polysaccharides on mice with exercise-induced fatigue and explore its possible mechanism of action. MethodICR male mice screened by swimming training were randomly divided into a control group， model group， vitamin C group， and low， medium， and high dose groups of Epimedii Folium polysaccharides， with eight mice in each group. The exercise-induced fatigue model was established by weight-bearing swimming training in each group except for the control group. After two weeks of weight-bearing swimming， the Epimedii Folium polysaccharide groups were given 100， 200， 400 mg∙kg-1 of Epimedii Folium polysaccharides by gavage， and the vitamin C group was given 200 mg∙kg-1 of vitamin C by gavage. The control group and the model group were given equal amounts of saline for 14 d. At the end of the experimental period， the body mass of the mice in each group and the time of last swimming due to exhaustion were recorded. Serum urea nitrogen （BUN）， lactic acid （LA）， lactate dehydrogenase （LDH）， malondialdehyde （MDA）， superoxide dismutase （SOD）， glutathione peroxidation （GSH-Px）， myoglycogen （MG） in skeletal muscle， hepatic glycogen （HG） in the liver were detected by kits. Hematoxylin-eosin （HE） staining was used to observe the pathological changes in muscle tissue. Western blot was used to detect the protein expression of p38 mitogen-activated protein kinase （p38 MAPK）， phosphorylation （p）-p38 MAPK， extracellular signal-regulated kinase1/2 （ERK1/2）， nuclear factor-κB （NF-κB）， p-NF-κB， interleukin-1β （IL-1β）， and interleukin-6 （IL-6） in muscle tissue. The immunofluorescence （IF） method was used to detect the expression of tumor necrosis factor-α （TNF-α） in skeletal muscle tissue of mice in each group. ResultCompared with the control group， the body mass of mice in the model group decreased， and the time of last swimming due to exhaustion decreased （P<0.01）. In addition， there were significantly higher serum levels of the fatigue metabolites LA， LDH， BUN， and lipid peroxidation product MDA （P<0.01） and decreased levels of MG， HG， SOD， and GSH-Px （P<0.01）. The protein expressions of p-p38 MAPK， ERK1/2， p-NF-κB， IL-1β， IL-6， and TNF-α in skeletal muscle tissue were significantly higher than those of the control group （P<0.01）. Compared with the model group， the body mass and time of last swimming due to exhaustion of the mice in the low， medium， and high dose groups of Epimedii Folium polysaccharides and the vitamin C group were increased （P<0.05， P<0.01）， and the contents of LA， LDH， BUN， and MDA were significantly decreased （P<0.05， P<0.01）. The levels of MG， HG， SOD， and GSH-Px increased （P<0.05， P<0.01）， and the protein expression levels of p-p38 MAPK， ERK， p-NF-κB， IL-1β， IL-6， and TNF-α in skeletal muscle tissue decreased （P<0.05， P<0.01）. ConclusionEpimedii Folium polysaccharides can play a role in alleviating exercise-induced fatigue by inhibiting the p38 MARK/NF-κB signaling pathway， thereby reducing the accumulation of metabolites， improving the activity of antioxidant enzymes， increasing the glycogen content of the body， and reducing inflammation in skeletal muscle.

Analysis of Chemical Components and Tissue Distribution of Lujiao Formula Based on UPLC-Q-Orbitrap-MS / 中国实验方剂学杂志

ObjectiveTo qualitatively analyze the chemical constituents and their tissue distribution in Lujiao formula based on ultra performance liquid chromatography-quadrupole-electrostatic field orbitrap high resolution mass spectrometry（UPLC-Q-Orbitrap-MS）. MethodThe separation was performed on an ACQUITY UPLC® BEH C18 column（2.1 mm×100 mm， 1.7 μm） with the mobile phase of 0.1% formic acid aqueous solution（A）-methanol（B） in a gradient elution（0-2 min， 4%B； 2-6 min 4%-12%B； 6-38 min， 12%-70%B； 38-38.5 min， 70%B； 38.5-39 min， 70%-95%B； 39-43 min， 95%B； 43-43.1 min， 95%-4%B； 43.1-45 min， 4%B）， the flow rate was 0.3 mL·min-1 with the column temperature of 40 ℃ and the injection volume of 5 µL. The data were acquired by an electrospray ionization（ESI） in the full scanning mode of positive and negative ions， the scanning rang was set at m/z 100-1 500， the collision energies were 10， 20， 40 eV. Retention time， precise relative molecular mass and secondary mass spectrometry fragment ions were used to identify the compounds in Lujiao formula and analyze their tissue distribution， combing with self-established database and comparing with standard substances and published literature data. ResultA total of 260 compounds， including 156 flavonoids， 43 terpenoids， 18 coumarins， 13 organic acids， 7 phenylethanoids， 7 alkaloids and 16 others， were identified or hypothesized from Lujiao formula， 68 of which were identified by comparison with standard substances. And the results of tissue distribution showed that 100， 143， 129 and 126 prototype components were detected in blood， heart， liver and kidney， respectively. ConclusionThe chemical composition of Lujiao formula and their tissue distribution were systematic analyzed， which can provide reference for the quality control， clinical application， pharmacokinetics and pharmacodynamic material basis of Lujiao formula and its medicinal materials.

Screening Effective Sites of Momordicae Semen-Epimedii Folium and Anti-lung Cancer Mechanism of Its Prescription / 中国实验方剂学杂志

ObjectiveTo preliminarily confirm the effective anti-lung cancer sites of Momordicae Semen and Epimedii Folium and study their mechanism of action. MethodOn the basis of preliminary research， the extraction method of Momordicae Semen and Epimedii Folium was optimized and the effective parts were screened under the guidance of pharmacological effects. Different ethanol elution and water elution sites of Momordicae Semen and Epimedii Folium were obtained through adsorption and elution with D101 macroporous resin. The methylthiazolyldiphenyl-tetrazolium bromide （MTT） colorimetric assay was used to detect the effects of total drug extracts and different elution sites on the proliferation of various tumor cell lines， and to screen for the optimal elution site and tumor sensitive strains. Flow cytometry was used to detect the effect of the elution sites of Momordicae Semen and Epimedii Folium on intracellular reactive oxygen species （ROS） and apoptosis in A549 cells. Western blot was used to compare the expressions of tumor protein 53 （p53）， Bcl-2-associated X protein （Bax）， cysteinyl aspartate specific proteinase-3 and 9 （Caspase-3 and Caspase-9） proteins in A549 cells. ResultThe inhibitory effect of Momordicae Semen on the proliferation of A549 cells was better than the kernel of Momordicae Semen， with 50% inhibitory concentration （IC50） being （86.83±2.88） mg·L-1 and （95.10±18.13） mg·L-1， respectively. The effect of total extracts of Epimedii Folium on A549 anti proliferation IC50 value was （4.71±0.81） mg·L-1. The IC50 values of the 40%， 60%， and 80% ethanol and anhydrous ethanol eluted macroporous resins of the total extracts of Momordicae Semen and Epimedii Folium inhibiting A549 proliferation were （45.32±4.38）、 （14.95±0.73）、 （17.07±1.76）、 （14.46±2.35）、 （51.7±2.26）、 （12.37±0.67）、 （20.29±0.93）、 and （3.43±0.91） mg·L-1， respectively. Compared with the normal group， the 1∶1 combination of Momordicae Semen and Epimedii Folium inhibited A549 cell proliferation in a time-dependent and concentration-dependent manner. Compared with the normal group， 50 mg·L-1 of the combination of Momordicae Semen and Epimedii Folium significantly increased intracellular ROS expression （P<0.01）. Compared with the normal group， 12.5， 25， 50 mg·L-1 of the combination of Momordicae Semen and Epimedii Folium significantly increased the expression of A549 cell apoptosis （P<0.01）. Compared with the normal group， 25， 50 mg·L-1 of the combination of Momordicae Semen and Epimedii Folium significantly increased the expression of p53 in A549 cells （P<0.01）. Compared with the normal group， 12.5， 25， 50 mg·L-1 of the combination of Momordicae Semen and Epimedii Folium significantly increased the expression of Bax （P<0.01）. Compared with the normal group， 50 mg·L-1 of the combination of Momordicae Semen and Epimedii Folium significantly reduced the expressions of Caspase-3 and Caspase-9 （P<0.01）. ConclusionThe anti-tumor effect of Momordicae Semen is better than that of the kernel of Momordicae Semen. The anti-tumor substances of Momordicae Semen and Epimedii Folium mainly concentrate in the 60% ethanol to anhydrous ethanol elution site. A549 cells are sensitive to the 1∶1 combination of Momordicae Semen and Epimedii Folium， which can effectively inhibit the cell proliferation. The mechanism may be related to increasing the generation of ROS in A549 cells， promoting their apoptosis， increasing the expressions of apoptotic proteins such as p53 and Bax， and reducing the expressions of Caspase-3 and Caspase-9.

Identification of mixed medicinal powder of Epimedii Folium with different origins based on pyrosequencing technology / 药学学报

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.

Exploring the safe and rational use of Epimedii Folium based on the evolution of efficacy/toxicity records in ancient and modern literature / 药学学报

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.

Multidisciplinary strategies to enhance therapeutic effects of flavonoids from Epimedii Folium:Integration of herbal medicine,enzyme engineering,and nanotechnology / 药物分析学报

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.

Research progress on the biotransformation of flavonoid glycosides in Epimedii Folium / 中国药房

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.

Mechanism of Epimedii Folium in Treatment of Oligoasthenotspermia Based on Network Pharmacology and Molecular Docking / 中国实验方剂学杂志

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.

Mechanism of Epimedii Folium in Treatment of Oligoasthenotspermia Based on Network Pharmacology and Molecular Docking / 中国实验方剂学杂志

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.

Analysis of the spectrum-effect relationship of different processed products of Epimedii folium on cytotoxicity / 国际中医中药杂志

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.

Research progress on sex-hormone-like effects of epimedium / 国际中医中药杂志

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.

Active Constituents and Mechanism of Epimedii Folium Against Liver Cancer： A Review / 中国实验方剂学杂志

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.

Effect of active component compound of Epimedii Folium,Astragali Radix,and Puerariae Lobatae Radix on expression of ADAM17 in HT22 cells by mediating hepcidin / 中国中药杂志

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.

Material Basis and Mechanism of Epimedii Folium Against Osteoporosis Based on Spectrum-Effect Relationship and Network Pharmacology / 中国实验方剂学杂志

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.

Mechanisms analysis of the reinforcing effect of Epimedii Folium on kidney in the treatment of polycystic ovarian syndrome with network pharmacology / 国际中医中药杂志

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.

Network Pharmacology-based Analysis on Mechanism of Aconiti Lateralis Radix Praeparata and Epimedii Folium in Treatment of Chronic Heart Failure / 中国实验方剂学杂志

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^-1） alone and the prescription （10 mg·L^-1）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^-1 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.

Biomarkers and Target Mechanisms of Epimedii Folium Intervention on Breast Cancer Stem Cells Based on Chip Analysis and Network Pharmacology / 中国实验方剂学杂志

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.

Effect of Epimedii Folium Total Flavonoids on Angiogenesis of Ischemic Myocardium in Rats After Acute Myocardial Infarction / 中国实验方剂学杂志

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^-1·d^-1）， and TEF low-dose and high-dose groups （100 and 200 mg·kg^-1·d^-1）， 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.