Holistic quality evaluation method of Epimedii Folium based on NIR spectroscopy and chemometrics.

INTRODUCTION: There are some problems in the quality control of Epimedii Folium (leaves of Epimedium brevicornum Maxim.), such as the mixed use of Epimedii Folium from different harvesting periods and regions, incomplete quality evaluation, and time-consuming analysis methods. OBJECTIVE: Near-infrared (NIR) spectroscopy was conducted to establish a rapid overall quality evaluation method for Epimedii Folium. MATERIALS AND METHODS: Quantitative models of the total solid, moisture, total flavonoid, and flavonol glycoside (Epimedin A, Epimedin B, Epimedin C, Icariin) contents of Epimedii Folium were established by partial least squares regression (PLSR). The root mean square error (RMSE) and correlation coefficient (R) were used to evaluate the performance of models. The qualitative models of Epimedii Folium from different geographic origins and harvest periods were established based on K-nearest neighbor (KNN), back-propagation neural network (BPNN), and random forest (RF). Accuracy and Kappa values were used to evaluate the performance of models. A new multivariable signal conversion strategy was proposed, which combines NIR spectroscopy with the PLSR model to predict the absorbance values of retention time points in the high-performance liquid chromatography (HPLC) fingerprint to obtain the predicted HPLC fingerprint. The Pearson correlation coefficient and cosine coefficient were used to evaluate the similarity between real and predicted HPLC fingerprints. RESULTS: Qualitative models, quantitative models, and the similarity between real and predicted HPLC fingerprints are satisfactory. CONCLUSION: The method serves as a fast and green analytical quality evaluation method of Epimedii Folium and can replace traditional methods to achieve the overall quality evaluation of Epimedii Folium.

[Determination of 13 chemical components of Epimedii Folium in pharmacopoeia by UPLC method combined with quantitative analysis of multicomponents by single-marker].

The quantitative analysis of multicomponents by single-marker(QAMS) was established for 13 chemical components of Epimedii Folium, including neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuoside Ⅰ, so as to investigate the feasibility and accuracy of this method in evaluating the quality of Epimedii Folium materials from different origins and different varieties. Through the scientific and accurate investigation of the experimental method, the external standard method was used to determine the content of 13 chemical components in epimedium brevieornu. At the same time, icariin was used as the internal standard, and the relative correction factors of icariin with neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuoside Ⅰ were established, respectively. The contens of neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuosideⅠ in Epimedii Folium were calculated by QAMS. Finally, the difference between the measured value and the calculated value was compared to verify the accuracy and scientific nature of QAMS in the determination. The relative correction factor of each component had better repeatability, and there was no significant difference between the results of the external standard method and those of QAMS. With icariin as the internal standard, QAMS simultaneously determining neoglycolic acid, chlorogenic acid, cryo-chlorogenic acid, magnolidine, hypericin, epimedin A, epimedin B, epimedin C, icariin, baohuoside Ⅱ, sagittatoside A, icariin subside Ⅰ, and baohuoside Ⅰ can be used for quantitative analysis of Epimedii Folium.

Baohuoside I inhibits FXR signaling pathway to interfere with bile acid homeostasis via targeting ER α degradation.

Epimedii folium (EF) is an effective herbal medicine in osteoporosis treatment, but the clinical utilization of EF has been limited due to potential hepatotoxicity. The previous studies identified that baohuoside I (BI), the main active component of EF, was relevant to EF-induced liver injury. However, the mechanisms of BI causing direct injury to hepatocytes remain unclear. Here, we reveal that BI inhibits FXR-mediated signaling pathway via targeting estrogen receptor α (ER α), leading to the accumulation of bile acids (BAs). Targeted bile acid analyses show BI alters the BA composition and distribution, resulting in impaired BA homeostasis. Mechanistically, BI induces FXR-dependent hepatotoxicity at transcriptional level. Additionally, ER α is predicted to bind to the FXR promoter region based on transcription factor binding sites databases and we further demonstrate that ER α positively regulates FXR promoter activity and affects the expression of target genes involved in BA metabolism. Importantly, we discover that ER α and its mediated FXR transcription regulation might be involved in BI-induced liver injury via ligand-dependent ER α degradation. Collectively, our findings indicate that FXR is a newly discovered target gene of ER α mediated BI-induced liver injury, and suggest BI may be responsible for EF-induced liver injury.

Comparative pharmacokinetic study of nine bioactive components in osteoarthritis rat plasma using ultra-performance liquid chromatography-tandem mass spectrometry after single and combined oral administration of Epimedii Folium and Chuanxiong Rhizoma extracts.

The herb pair Epimedii Folium-Chuanxiong Rhizoma (EF-CR), derived from the classical traditional Chinese medicine 'Xian Ling Pi San', has a distinctive compatibility therapeutic profile and is clinically safe and effective. This study aimed to investigate and compare the pharmacokinetic characteristics of nine analytes in osteoarthritis (OA) rat plasma after the oral administration of EF, CR or a combination of these two herbs. We developed an ultra-performance liquid chromatography method coupled with quadrupole linear ion-trap mass spectrometry to simultaneously quantify and assess the pharmacokinetics of icariin, epimedin A, epimedin B, epimedin C, icariside I, icariside II, ferulic acid, ligustilide and senkyunolide A of the EF-CR pair in the plasma of osteoarthritic rats. The pharmacokinetic parameters showed that the absorption of multiple components was significantly enhanced and residence time was prolonged in the EF-CR group (P < 0.05) compared to the single-herb group. These parameters revealed that the combination of EF and CR exhibited synergistic effects of the nine bioactive components, suggesting the potential application of the EF-CR combination for the treatment of OA.

Screening of superior anti-osteoporotic flavonoids from Epimedii Folium with dual effects of reversing iron overload and promoting osteogenesis.

Iron overload is a risk factor for postmenopausal osteoporosis (PMOP) and lowering iron levels to regulate the labile plasma iron is the preferred therapy. Icariin (ICA), baohuoside I (BHS) and icaritin (ICT) are three flavonoids obtained from Epimedii Folium that are efficient in facilitating osteogenesis. In this study, an active flavonoid with dual effects of reversing iron overload and promoting osteogenesis was screened based on pharmacokinetics, iron complexation properties and the potential to downregulate iron overload, reversing PMOP. As a result, the in vivo absorption of three compounds was ICA > ICT > BHS, while the exposure in muscle and bone was BHS > ICT > ICA. In vitro complexation showed that only ICT complexed with Fe (III) at a 1:1 ratio on 3-OH and the ICT-Fe (III) complex (m/z 424.3750) was identified by UPLC-Q-TOF-MS. In vivo dynamic detection also showed that the concentration of ICT-Fe (III) complexes varied with the concentration of ICT in plasma. The behavioral blunting and bone loss in zebrafish induced by Fe (III) were significantly reversed by ICT in a dose-dependent manner. Pharmacokinetic-pharmacodynamic analysis showed that ICT was negatively correlated with serum ferritin and positively correlated with osteogenic markers including alkaline phosphatase, osteocalcin and osteoprotegerin. Bone loss in ovariectomized rats was significantly altered after ICT intervention, with reduced serum ferritin levels and improved osteogenic marker levels. These results demonstrated that ICT had favorable musculoskeletal penetration and iron complexation capability to shrink labile plasma iron, showing superior performance in anti-PMOP through the dual effects of reversing iron overload and promoting osteogenesis.

Effects of Bushen Yiyuan recipe on testosterone synthesis in Leydig cells of rats with exercise-induced low serum testosterone levels.

CONTEXT: Bushen Yiyuan recipe (BYR) is an effective Chinese prescription with antifatigue and antioxidation effects. OBJECTIVE: The effects of BYR on testosterone synthesis in rat Leydig cells with exercise-induced low serum testosterone levels (EILST) are assessed. MATERIALS AND METHODS: Thirty-two Sprague-Dawley rats were chronically trained for 6 weeks to establish an EILST model. EILST rats were divided into model (physiological saline), EFE (700 mg/kg ethanol extract of Epimedii folium, the dried leaves of Epimedium brevicornu Maxim [Berberidaceae]), and BYR groups (350 and 700 mg/kg) for 6 weeks. Expression of HMG-CoA, LDL-R, SR-BI, STAR and CYP11A1 were quantified by RT qPCR and Western blots. RESULTS: Compared with the model group (115.52 ± 13.05 µg/dL; 67.83 ± 14.29; 0.32 ± 0.04; 0.33 ± 0.02; 0.38 ± 0.01), serum testosterone, testosterone/cortisol ratio, HMG-CoA, STAR and CYP11A1 relative protein expression significantly increased in low-dose BYR (210.60 ± 5.08 µg/dL; 119.38 ± 13.02; 0.47 ± 0.01; 0.46 ± 0.03; 0.46 ± 0.02), high-dose BYR (220.57 ± 14.71 µg/dL; 124.26 ± 14.79; 0.49 ± 0.02; 0.42 ± 0.03; 0.51 ± 0.02), and EFE groups (206.83 ± 5.54 µg/dL; 119.53 ± 25.04; 0.45 ± 0.02; 0.42 ± 0.02; 0.41 ± 0.02) (all p < 0.01, except for CYP11A1 in EFE group). HMG-CoA, STAR and CYP11A1 mRNA relative expression significantly increased in low-dose and high-dose BYR group compared to model group (all p < 0.01). CONCLUSIONS: BYR affects endogenous cholesterol synthesis and testosterone synthesis to prevent and treat EILST levels in rats. It can improve the body's sports ability.

[Efficacy and mechanism of low glycoside from Epimedii Folium flavonoids on retinoic acid-induced osteoporosis in rats].

In this study, the secondary osteoporosis model was induced by oral administration of retinoic acid for two weeks in SD male rats. The efficacy and mechanism of LG on secondary osteoporosis in rats were explored through the bone morphogenetic protein 2(BMP-2)/Runt-related transcription factor 2(Runx2)/Osterix signaling pathway. With Xianling Gubao Capsules(XLGB) as the positive control, three dose groups of low glycoside from Epimedii Folium flavonoids(LG), i.e., low-dose group(LG-L), medium-dose group(LG-M), and high-dose group(LG-H), were set up. After modeling, the rats in each group were treated correspondingly by gavage for eight weeks. The action target of LG in the treatment of secondary osteoporosis in rats was analyzed by measuring the body weight and the organ indexes of rats including heart index and testis index. The efficacy of LG was characterized by the pathological changes of the femur, the microstructural parameters of the trabecular bone, and the biomechanical properties of femoral tissues in rats. The mechanism of LG was explored by measuring the relevant biochemical indexes and the changes in BMP-2, Runx2, and Osterix content in rats with secondary osteoporosis. The results showed that the action target of LG in the treatment of secondary osteoporosis in rats was the testis. LG can improve the bone loss of the femur, increase the number and thickness of the trabecular bone, reduce the porosity and separation of the trabecular bone, potentiate the resistance of bone to deformation and destruction, up-regulate the serum content of Ca, P, aminoterminal propeptide of type Ⅰ procollagen(PINP), and osteocalcin(OC), promote bone matrix calcification and the expression of BMP-2, Runx2, and Osterix proteins, and accelerate bone formation, thereby reducing the risk of fractures, and ultimately exerting anti-secondary osteoporosis efficacy.

[Composition and immunomodulatory activity of neutral and acidic polysaccharides isolated from Epimedii Folium].

Epimedii Folium possesses many pharmacological activities including immunomodulation, anti-oxidation, and anti-tumor. Polysaccharides are the main components of Epimedii Folium, and their activities are closely related to the structure. The present study isolated a neutral polysaccharide(EPS-1-1) and an acidic polysaccharide(EPS-2-1) from the aqueous extract of Epimedii Folium through DEAE-52 cellulose anion-exchange chromatography and Sephadex G-100. The structures were characterized by chemical composition analysis, high-performance gel permeation chromatography(HPGPC), Fourier-transform infrared spectrometry(FT-IR), 1-phenyl-3-methyl-5-pyrazolone(PMP) derivatization, scanning electron microscopy(SEM), Congo red test, etc. The immunomodulatory activity of polysaccharides in vitro was determined by investigating the effects on the maturation of bone marrow-derived dendritic cells(BMDCs) and the release of inflammatory cytokines. According to the structural characterization analysis, EPS-1-1 was composed of fructose(Fuc), mannose(Man), ribose(Rib), rhamnose(Rha), glucose(Glc), galactose(Gal), xylose(Xyl), and arabinose(Ara) at 1.90∶0.67∶0.05∶0.08∶3.29∶1.51∶0.05∶0.37(molar ratio), while EPS-2-1 was mainly composed of Fuc, Man, Rha, glucuronic acid(GlcA), galacturonic acid(GalA), Glc, Gal, Xyl, and Ara at 5.25∶0.18∶0.32∶0.13∶1.14∶0.16∶0.55∶0.08∶0.2. EPS-1-1 and EPS-2-1 could promote the maturation and function of BMDCs through up-regulating the expression of MHC-Ⅱ, CD86, CD80, and CD40, and increasing the levels of inflammatory cytokines(IL-6, IL-12, and TNF-α) in vitro experiments, which suggested that EPS-1-1 and EPS-2-1 possessed good immunomodulatory activity.

[Preparation of microemulsion gel loading enriched ingredients of Epimedii Folium and its pharmacodynamics evaluation].

On the basis of previous studies, this study prepared and evaluated microemulsion gel loading enriched ingredients of Epimedii Folium and investigated its protective effect against peripheral nervous system damage caused by chemotherapeutics. The preparation method and the type and dosage of the matrix were investigated from rheology, preparation difficulty, and drug loading. Then the optimal prescription was determined and the microemulsion gel loading enriched ingredients of Epimedii Folium was prepared. The in vitro release and transdermal behaviors of the gel were investigated in the Franz diffusion cell with epimedin A1,A,B,C, and icariin as evaluation indicators. The oxaliplatin-induced peripheral neuropathy(OIPN) model was established in Wistar rats. The protective effect of the microemulsion gel loading enriched ingredients of Epimedii Folium against peripheral nervous system damage caused by chemotherapeutics was evaluated by behavioral measurement after drug administration and histopathological examination of dorsal root ganglia and sciatic nerve. The preparation process of the microemulsion gel loading enriched ingredients of Epimedii Folium was stable, and the release of the five components was consistent with the Hixson-Crowell cube root law. Behavioral indicators intuitively showed that the drug could effectively relieve mechanical allodynia caused by oxaliplatin. The histopathological examination showed that the drug can improve neuron damage in the dorsal root ganglia, axon degeneration, and demyelination caused by oxaliplatin. Therefore, the preparation process of the microemulsion gel loading enriched ingredients of Epimedii Folium is feasible, which can achieve stable drug release. It has a certain therapeutic effect on chemotherapy-induced peripheral neuropathy(CIPN).

Icariside I specifically facilitates ATP or nigericin-induced NLRP3 inflammasome activation and causes idiosyncratic hepatotoxicity.

BACKGROUND: Epimedii Folium (EF) is commonly used for treating bone fractures and joint diseases, but the potential hepatotoxicity of EF limits its clinical application. Our previous study confirms that EF could lead to idiosyncratic drug-induced liver injury (IDILI) and hepatocyte apoptosis, but the mechanism remains unknown. Studies have shown that NLRP3 inflammasome plays an important role in the development of various inflammatory diseases such as IDILI. Specific stimulus-induced NLRP3 inflammasome activation may has been a key strategy for lead to liver injury. Therefore, main compounds derived from EF were chosen to test whether the ingredients in EF could activate the NLRP3 inflammasome and to induce IDILI. METHODS: Bone-marrow-derived macrophages (BMDMs) were treated with Icariside I, and then stimulated with inflammasome stimuli and assayed for the production of caspase-1 and interleukin 1ß (IL-1ß) and the release of lactate dehydrogenase (LDH). Determination of intracellular potassium, ASC oligomerization as well as reactive oxygen species (ROS) production were used to evaluate the stimulative mechanism of Icariside I on inflammasome activation. Mouse models of NLRP3 diseases were used to test whether Icariside I has hepatocyte apoptosis effects and promoted NLRP3 inflammasome activation in vivo. RESULTS: Icariside I specifically enhances NLRP3 inflammasome activation triggered by ATP or nigericin but not SiO2, poly(I:C) or cytosolic LPS. Additionally, Icariside I does not alter the activation of NLRC4 and AIM2 inflammasomes. Mechanically, Icariside I alone does not induce mitochondrial reactive oxygen species (mtROS), which is one of the critical upstream events of NLRP3 inflammasome activation; however, Icariside I increases mtROS production induced by ATP or nigericin but not SiO2. Importantly, Icariside I leads to liver injury and NLRP3 inflammasome activation in an LPS-mediated susceptibility mouse model of IDILI, but the effect of Icariside I is absent in the LPS-mediated mouse model pretreated with MCC950, which is used to mimic knockdown of NLRP3 inflammasome activation. CONCLUSIONS: Our study reveals that Icariside I specifically facilitates ATP or nigericin-induced NLRP3 inflammasome activation and causes idiosyncratic hepatotoxicity. The findings suggest that Icariside I or EF should be avoided in patients with diseases related to ATP or nigericin-induced NLRP3 inflammasome activation, which may be risk factors for IDILI. Video abstract.

[Potential hepatotoxic compounds and mechanisms of Epimedii Folium based on network toxicology and cell experimental validation].

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.

[Effect of active component compound of Epimedii Folium,Astragali Radix,and Puerariae Lobatae Radix on expression of ADAM17 in HT22 cells by mediating hepcidin].

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 ß 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, Aß25-35 induction(Aß) group, hepcidin-siRNA(siRNA) group, Aß25-35 + hepcidin-siRNA(Aß + siRNA) group, Aß25-35+YHG(Aß+YHG) group, hepcidin-siRNA+YHG(siRNA+YHG) group, Aß25-35+hepcidin-siRNA+YHG(Aß+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 Aß group, siRNA group, and Aß+siRNA group than in the control group(P<0.05) and the expression was lower in the Aß+siRNA group(P<0.05) and higher in the Aß+YHG group(P<0.05) than in the Aß group. Moreover, the ADAM17 protein expression was lower in the Aß+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 Aß+siRNA+YHG group than in the Aß+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.

[Research and verification of quality evaluation method of Epimedii Folium based on quantitative analysis of multi-components by single marker].

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.

[Screening combination ratio and exploring mechanism of Momordicae Semen and Epimedii Folium].

The aim of this paper was to obtain low toxicity and high efficiency anti-tumor Chinese medicine through screening the combination ratios of Momordicae Semen and Epimedii Folium, and to explore the anti-tumor mechanism of the combination of two drugs by observing their effect on apoptosis-related proteins in cancer cells. Methyl thiazolyl tetrazolium(MTT) assay was used to observe the effect of drug combination on the proliferation of tumor cells from different tissue sources. The effects of the combination of the two drugs on tumor cells were analyzed by Compusyn software. Plate cloning assay was used to observe the effect of combination of these two drugs on the proliferation of A549 cells in vitro. The expression of reactive oxygen species(ROS) and apoptotic proteins p53, Bcl-2 and Bax were compared by using ROS kit and Western blot. Lewis lung cancer model was used to observe the anti-tumor effect of drugs in vivo. The results showed that the anti-tumor effect of their ethanol extract was more significant than that of water extract, and the anti-proliferation effect was strongest when the ratio was 1∶1(P<0.05). Compusyn analysis showed that the combination of the two drugs had synergistic effect. Further studies showed that after combined use, the number of clonogen formation in A549 cells was significantly reduced(P<0.01); ROS production was increased; the expression of apoptosis-related protein p53 was up-regulated, and the ratio of Bcl-2/Bax was decreased. In vivo animal study showed that the tumor inhibition rate was 53.06%(P<0.05) in the high dose group. As compared with the single use of the two drugs, the combination of the two drugs had more significant anti-proliferative effect on tumors, and the optimum ratio was 1∶1. The combination of the two drugs at a ratio of 1∶1 inhibited the proliferation of various tumor cells, and had no significant effect on normal liver cells LO2 when compared with other ratios. Therefore, it can be preliminarily inferred that the combination of the two drugs may have the effect of synergism and detoxification. Further studies showed that the combination of the two drugs can significantly inhibit the proliferation of A549 cells, and its mechanism may be related to the activation of endogenous apoptotic pathway. In vivo experiments also showed that the tumor inhibition rate increased with the increase of drug concentration.

[Ethanol extraction technology of Epimedii Folium and protective effect of ethanol extract on chondrocyte].

Uniform design-comprehensive scoring method was used to investigate the effects of ethanol dosage, ethanol concentration and extraction time, based on the evaluation index from transfer rates of epimedin A, epimedin B, epimedin C, icariin and baohuoside Ⅰ, which are the main active components in Epimedii Folium. Furthermore, the optimum conditions for the ethanol extraction process were determined by multiple linear stepwise regression and empirical test. Then, the ethanol extract of Epimedii Folium prepared according to the optimized technological conditions was used to intervene the injury model of chondrocyte induced by interleukin-1 beta(IL-1ß). Annexin V-FITC/PI staining flow cytometry was used to detect the apoptotic rate of chondrocyte and analyze the effect of ethanol extract of Epimedii Folium on chondrocyte injury model. The optimum conditions of ethanol extraction were as follows. Crude powder of Epimedii Folium was added with 18 times of 70% ethanol solution, and extracted for twice in the refluxing process, for 60 minutes each time. Under the conditions, the extraction rates of the above five active components were 94.21%, 94.76%, 93.85%, 96.17% and 96.85%, respectively. The optimized ethanol extraction process of Epimedii Folium was reasonable, feasible and reproducible. This ethanol extract could significantly reduce the early apoptotic rate, late apoptotic and necrotic rate, total apoptotic rate(P<0.05 or P<0.01) of chondrocyte injury model induced by IL-1ß, suggesting that the ethanol extract of Epimedii Folium can inhibit the apoptosis of chondrocytes induced by IL-1ß to a certain extent, which lays a foundation for further study on its prevention and treatment of osteoarthritis.

[Study on mechanism of "Epimedii Folium-Paeoniae Radix Alba" in treatment of lumbar disc herniation based on network pharmacology].

The aim of this paper was to investigate the key targets and mechanism of "Epimedii Folium-Paeoniae Radix Alba" in the treatment of lumbar disc herniation by means of network pharmacology. The currently recognized databases and analysis software at home and abroad were used to construct the network from drugs and diseases. The chemical components of Epimedii Folium and Paeo-niae Radix Alba were collected by using databases such as TCMSP, while their active components were determined and the action targets were predicted according to threshold screening and literature reports. The genes for lumbar disc herniation were collected by using GeneCards, OMIM, and DisGeNET databases. The drug targets were mapped to disease targets, and protein interaction network analysis for key targets, GO function enrichment analysis and KEGG signaling pathway enrichment analysis were performed. Finally, 23 active components of Epimedium Folium and 13 active components of Paeoniae Radix Alba were determined, and a total of 624 drug targets were obtained. After standardization, 214 drug targets were obtained. In addition, 306, 2 and 5 related targets of lumbar disc herniation were collected from GeneCards, OMIM, and DisGeNET database, respectively, and a total of 293 disease targets were obtained after deduplication. After the mapping of drug target and disease target, 44 common targets were obtained. PPI protein interaction network analysis showed that IL-6, TNF, AKT1, MAPK1, and VEGFA may be the core targets for the treatment of lumbar disc herniation. GO enrichment analysis identified 56 items(P<0.05), among which biological processes mainly included immune response, apoptosis, etc.; cell components mainly included extracellular space, extracellular region, etc.; molecular functions mainly included cytokine activity, metallopeptidase activity and so on. Through KEGG pathway enrichment analysis, 91 signaling pathways related to inflammation, metabolism, and senescence were identified, mainly including IL-17 signaling pathway and TNF signaling pathway and so on. "Epimedii Folium-Paeoniae Radix Alba" showed the characteristics of multi-channel and multi-target for the treatment of lumbar disc herniation. This study preliminarily explored the key targets for its role and the biological processes and signaling pathways involved. It was found that it may play a therapeutic role by affecting inflammation and immune regulation, which laid the foundation for further experimental verification.

[Molecular mechanism of Epimedii Folium in treatment of osteoporosis based on network pharmacology].

Osteoporosis is a systematic bone disease,characterized by deterioration in bone mass or micro-architecture,and increasing risk of fragility and fractures. With the development of aging problems,osteoporosis has been a global health problem. At present,due to the undesirable side effects of synthetic osteoporosis inhibitors,more efforts are made in treatment of osteoporosis by traditional Chinese medicine and its prescriptions. Epimedii Folium,one of the most common herbs for osteoporosis,has attracted great attentions worldwide.In this study,network pharmacology was employed to analyze the active components and potential molecular mechanism of Epimedii Folium on osteoporosis. Component-target network analysis showed that those with higher molecular network degree were flavonoids,with estrogen-like activity,antioxidation and free radical-scavenging activities,playing certain roles in preventing and treating osteoporosis. On the other hand,the targets with high degree were mostly related with sex hormone,osteoclast differentiation,bone matrix degradation,and reactive oxygen species in drug-target network. Multiple components of Epimedii Folium could be interacted with these targets. This study shows that Epimedium could prevent and treat osteoporosis through multiple active ingredients acting on multiple targets.

[Discussion on safety evaluation and risk control measures of Epimedii Folium].

Epimedii Folium,a commonly used traditional Chinese medicine,has the effect of tonifying kidney Yang,strengthening bones and treating rheumatism. However,in recent years,the number of reports on adverse reactions of Epimedii Folium and its Chinese patent medicines such as Xianling Gubao Capsules and Zhuanggu Guanjie Pills has been gradually increased,and the toxicity of Epimedii Folium has attracted more and more attention. In this article,the ancient and modern literature on Epimedii Folium was traced through a comprehensive and systematic literature analysis method. According to the 2015 edition of the Chinese Pharmacopoeia,Epimedii Folium refers to the dried leaves of Epimedii Folium brevicomu,E. sugittutum,E. pubescens or E. koreuuum. The Chinese Pharmacopoeia also includes E. wushanense of Wushan Epimedium,which is the same plant variety as Epimedium. The study showed that there were differences in the geographical distribution,composition and toxicity among five species of Epimedium. This paper also explained the toxicity mechanism as well as efficacy enhancing and toxicity reducing effects of Epimedii Folium,and reported its related adverse reaction cases. Through a retrospective comparative study on the toxicity of the modern Chinese patent medicines Xianling Gubao Capsules and Zhuanggu Guanjie Pills containing Epimedii Folium,it was believed that Epimedii Folium had cardiovascular system toxicity,neurotoxicity,hepatotoxicity,long-term toxicity,acute toxicity,genotoxicity and special toxicity; its safe medication factors included patient syndrome,doctor factors,drug factors,processing and compatibility factors. Meanwhile,strategies were proposed to improve patient safety medication awareness,standardize Epimedii Folium varieties and quality supervision,and the toxicity of Epimedii Folium was studied,hoping to draw attention from scholars to the safety of Epimedii Folium,improve the safe use of Epimedii Folium,and prevent adverse reactions.

[Change of icariin content in five Epimedium species by heating process].

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

[Research actuality and quality-influencing factor of Epimedii Folium].

Epimedii Folium has a long history in China as a common traditional Chinese medicine. Key factors of Epimedii Folium quality were summarized based on ancient literatures, Chinese Pharmacopoeias and modern research in different period of history. The main reason for unqualified Epimedii Folium is unstable icariin. Therefore, it's suggested that: the precondition of the quality control of epimedium is to find the proper quality marker. It's suggested that the medicinal parts should be reverted to "dry whole plant overground" to solve Epimedium resource shortage problem. In addition, it is necessary to strengthen the standardized cultivation, so as to ensure germplasm, production area, and producing method to guarantee the quality of Epimedium Folium. In the drying method, it is recommended to change "dry in the sun or shade" to "dry", namely dry in the sun, shade or drier, in order to provide a new method to improve the quality control and quality standard of Epimedii Folium.