Potential hepatotoxic compounds and mechanisms of Epimedii Folium based on network toxicology and cell experimental validation / 中国中药杂志

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 icaritin on proliferation and apoptosis of human ovarian cancer cells SKOV3 / 中国中药杂志

Based on the PI3K/Akt signaling pathway, this study aimed to observe the proliferation and apoptosis of ovarian cancer SKOV3 cells at different concentrations of icaritin, in order to explore the possible molecular mechanisms. The research object was ovarian cancer SKOV3 cells. The cells were divided into the control group and icaritin groups(5, 10, 20 μmol·L~(-1)), and administrated with drugs for 48 hours. The cell counting kit-8(CCK-8)assay was used to detect the inhibitory effect of icaritin on the proliferation of ovarian cancer SKOV3 cells. The proliferation ability of the SKOV3 cells was detected by EdU assay. Hoechst 33342 fluorescence staining was used to observe the apoptotic morphology of SKOV3 cells in each group. The distribution of cell cycle and the apoptosis rate of each group were detected by flow cytometry. Quantitative Real-time PCR was used to detect mRNA expressions of PTEN, PI3K, Akt in each group of cells. Protein expressions of PTEN, PI3K, Akt and p-Akt were measured by Western blot. The results showed that the cell inhibition rates of icaritin groups were significantly increased compared with the control group(P<0.05). The rates of EdU-positive cells of icaritin groups were significantly decreased(P<0.05). SKOV3 cells in icaritin groups showed morphological changes of apoptosis. Apoptosis rates of icaritin groups were significantly increased(P<0.05). The proportions of cells in G_0/G_1 phase of icaritin groups were decreased(P<0.05), while the proportions of S phase cells were increased(P<0.05). The gene and protein expressions of PTEN in icaritin groups were elevated(P<0.05). The gene expressions of PI3K and Akt in icaritin groups were down-regulated(P<0.05). The protein expression of PI3K and p-Akt in icaritin groups were reduced(P<0.05). These results indicated that icarin may inhibit the proliferation of ovarian cancer SKOV3 cells in vitro, induce cell apoptosis and affect the cycle distribution of cells by inhibiting the PI3K/Akt signaling pathway.

Inhibition of exosomes-mediated melanoma metastasis by icaritin / 药学学报

This study investigated the mechanism by which icaritin (ICT) inhibits exosomes-induced lung metastasis of B16BL6 mouse melanoma cells. The culture supernatant of B16BL6 cells was collected for extraction of exosomes by ultracentrifugation and their characterization by transmission electron microscopy and Western blotting. Exosomal protein was quantified by BCA. A wound-healing assay was used to determine the effect of ICT on the migratory ability of B16BL6 cells induced by exosomes. After establishing an experimental melanoma lung metastasis model in C57BL/6 mice, we used H&E staining to study the ability of ICT to inhibit exosomes-induced melanoma metastasis. Animal experiments were approved by the Ethics Committee of Nanjing University of Chinese Medicine. ELISA and immunofluorescence were used to detect pro-inflammatory factors interleukin 6 (IL-6), S100 calcium-binding protein A8/A9 complex (S100A8/A9), serum amyloid A (SAA) and fibronectin in metastatic tumors. The expression of metastatic tissue-related proteins stimulator of interferon gene (STING), phospho-STING (p-STING), TANK-binding kinase 1 (TBK1) and phospho-TBK1 (p-TBK1) was detected by immunohistochemistry or Western blotting. The results showed that the particle size of exosomes was 149.33 ± 2.68 nm, the polydispersity index (PDI) was 0.192 ± 0.02, the zeta potential was -32.22 ± 0.50 mV, and the particles had classic tea tray-like membrane structure under TEM. The protein concentration of exosomes was measured to be 838.66 ± 62.14 μg·mL-1. The results of the cell scratch test showed that ICT can inhibit exosomes-induced migration of B16BL6 cells at a concentration of 5, 10, and 20 μmol·L-1. In vivo experimental results also showed that ICT can inhibit exosomes-induced metastasis of melanoma to the lungs and can significantly inhibit the expression of pro-inflammatory factors S100A8/A9, SAA and IL-6 in lung tissue, and inhibit the expression of p-STING and p-TBK1 in metastatic lung tissue. Taken together, these results indicated that ICT can significantly inhibit exosomes-induced tumor metastasis, and the inhibition is related to the inactivation of STING in metastatic foci.

Effect of Icaritin on Proliferation， Apoptosis， Migration and Invasion of Human Ovarian Cancer A2780 Cells / 中国实验方剂学杂志

Objective:To study the effect of icaritin on the proliferation， apoptosis， migration and invasion of human epithelial ovarian cancer A2780 cells and the inhibitory mechanism of icaritin against cell invasion and migration via the regulation of epithelial-mesenchymal transformation （EMT）-related molecule expression. Method:A2780 cells were divided into the blank control group and low-， medium-， and high-dose （5， 10， 20 μmol·L^-1） icaritin groups and received the corresponding inventions for 48 h. Cell proliferation and viability were detected using the cell counting kit-8 （CCK-8）. The cellular proliferation inhibition and apoptosis rates were assayed by flow cytometry. The cell invasion and migration were observed in Scratch test and transwell test， followed by the calculation of wound healing rate and migration rate. The protein and mRNA expression levels of EMT-related molecules including E-cadherin， N-cadherin， and Vimentin and tumor invasion and migration-related molecule matrix metalloproteinase-9 （MMP-9） were measured by Western blot and real-time polymerase chain reaction （Real-time PCR）. Result:As revealed by CCK-8 assay and flow cytometry， compared with the blank control group， the icaritin groups all exhibited elevated proliferation inhibition rate （<italic>P</italic><0.01） and apoptosis rate （<italic>P</italic><0.05）. According to the Scratch test and transwell test， compared with the blank control group， the icaritin groups displayed weakened invasion and migration ability and decreased number and rate of cell invasion and migration （<italic>P</italic><0.05）. Western blot and Real-time PCR results showed that the protein and mRNA expression levels of N-cadherin， MMP-9 and Vimentin in each icaritin group were down-regulated as compared with those in the blank control group， while the expression of E-cadherin was up-regulated. Conclusion:Icaritin inhibits the proliferation and promotes the apoptosis of human ovarian cancer A2780 cells， and it inhibits the invasion and migration of A2780 cells possibly by regulating the expression of EMT-related molecules.

Study on the mechanism of proliferation-inhibiting effect of icaritin on hepatoma cells via regulating miRNA-329 and miRNA-1236 / 肿瘤研究与临床

Objective:To investigate the molecular mechanism of proliferation-inhibiting effect of icaritin on hepatoma cells via regulating miRNA-329 (miR-329) and miRNA-1236 (miR-1236).Methods:Hepatoma cell line HepG2 was treated with icaritin at different concentrations (2.5, 5.0, 10.0, 20.0, 40.0 μmol/L), and the control group only added dimethyl sulfoxide (DMSO). The half inhibitory concentration ( IC50) of icaritin on HepG2 cells and cell proliferation rate were detected by CCK-8 after cultured for 36 h. HepG2 cells were treated with 400 μg/L alpha fetoprotein (AFP). After cultured for 0, 12, 24, 36, 48 and 60 h, the effect of AFP on the proliferation of HepG2 cells was detected by CCK-8 method. AFP-3'UTR reporter plasmid pmirGLO-AFP-3'UTR plasmid was constructed, pmirGLO blank vector plasmid, pmirGLO-AFP-3'UTR plasmid, miR-329 or miR-1236 mimics or inhibitors, control plasmid of mimics (NC), control plasmid of inhibitors (INC) were respectively co-transfected with HepG2 cells, and the effect of miR-329 and miR-1236 on the luciferase activity was detected by dual-luciferase reporter assay after cultured for 24 h. Western blot and real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) were used to detect the effects of icaritin on the expressions of AFP, miR-329 and miR-1236 in HepG2 cells. HepG2 cells were respectively transfected with mimics and inhibitors of miR-329 and miR-1236 to detect the effects of miR-329 and miR-1236 on the expression of AFP. Results:The cell proliferation rates of 2.5, 5.0, 10.0, 20.0, 40.0 μmol/L icaritin group and control group were (80.4±2.3)%, (73.2±1.6)%, (51.7±3.3)%, (38.2±4.6)%, (29.5±4.3)%, and (94.0±2.9)%, respectively, and the difference was statistically significant ( F = 75.65, P < 0.01); the differences in the proliferation rate of HepG2 cells between different concentrations of icaritin group and control group were statistically significant (all P < 0.01). The IC50 of icaritin on HepG2 cells was 10 μmol/L. The relative expressions of AFP mRNA in 2.5, 5.0, 10.0, 20.0, 40.0 μmol/L icaritin group and control group were 0.83±0.06, 0.69±0.02, 0.53±0.07, 0.45±0.01, 0.33±0.07, and 1.00±0.01, respectively, and the difference was statistically significant ( F = 42.67, P < 0.01); the differences in the relative expressions of AFP mRNA between different concentrations of icaritin group and control group were statistically significant (all P < 0.01). HepG2 cells were treated by 400 μg /L AFP for 0, 12, 24, 36, 48 and 60 h, and the cell proliferation rates were (102±5)%, (138±13)%, (186±24)%, (260±12)%, (311±15)%, and (348±25)%, respectively, and the difference was statistically significant ( F = 27.483, P < 0.01); the differences in the cell proliferation rate between different time of AFP treatment and 0 h were statistically significant (all P < 0.01). Compared with the control group, different concentrations of icaritin can promote the expression of miR-329 and miR-1236 (all P < 0.01). After co-transfection of miR-329 and miR-1236 mimics and AFP-3'UTR, the luciferase activity decreased by about 40%; after co-transfection of miR-329 and miR-1236 inhibitors and AFP-3'UTR, the luciferase activity increased about 1.5 times. Both miR-329 and miR-1236 can reduce the expression levels of AFP protein and mRNA (both P < 0.05). Conclusion:Icaritin can promote the binding of miR-219, miR-1236 and AFP-3'UTR by promoting the expression of miR-329 and miR-1236, inhibit the stability and translation activity of AFP mRNA, inhibit the expression of AFP, and thus inhibit the proliferation of hepatoma cells in vitro.

Development of preparation process of icaritin-coix seed oil microemulsion based on quality by design concept / 中国中药杂志

To optimize the formulation and preparation process of icaritin-coix seed oil microemulsion(IC-MEs) based on quality by design(QbD) concept. IC-MEs were prepared by water titration. Firstly, the risk factors that may affect the quality of IC-MEs were evaluated. Then Plackett-Burman design was used to screen out prescription factors and process parameters that had a significant effect on the indicators. Finally, Box-Behnken design was used to optimize the prescription ratio of IC-MEs. Through the risk assessment and Plackett-Burman design, three formulation factors [drug loading efficiency, the ratio of mixed-oil(coix seed oil-Glycerol tributyrate) to mixed-surfactant(HS15-RH40) and water addition] were determined as the key factors affecting IC-MEs. The regression model established by Box-Behnken design had a good predictability. The optimal formula was as following: the drug loading efficiency of 0.92%, the ratio of mixed-oil(coix seed oil-glycerol tributyrate) to mixed-surfactant(HS15-RH40) of 4∶6, and the water addition of 5.7 mL. According to this prescription, IC-MEs were prepared, and its encapsulation efficiency after 1 week was 92.45%±1.00%. Therefore, the stability of IC-MEs could be improved by optimizing prescription and process parameters of IC-MEs based on the QbD concept, which can provide certain reference value for the future development of IC-MEs.

Research progress on pharmacology effects and new drug delivery system of icaritin / 中草药

Icaritin is one of the major bioactive compounds of Epimedii Folium. In recent years, scientific and pharmacological studies have shown that icaritin possesses broad therapeutic effects, especially in anti-tumor, enhancing osteoprotective, cardioprotective, neuro-protective function. However, its clinical application was limited by poor water solubility, poor oral absorption and low bioavailability. New drug delivery system has great application prospect in improving solubility of icaritin, oral absorption and bioavailability. Therefore, the current review paper aims to summarize the pharmacological effects of icaritin and briefly introduce some new drug delivery of icaritin, to provide theoretical basis for its clinical application and new drug development.

Effect of trifluoro-icaritin on protein levels related to Akt/mTOR signaling pathway and autophagy in myocardial ischemia/reperfusion injury rats / 中国药理学与毒理学杂志

OBJECTIVE: To investigate the effect of trifluoro-icaritin (ICTF) on myocardial ischemia/reperfusion injury (MI/RI) in rats and to explore whether it plays a role in regulating autophagy through the serine/threonine kinase/mammalian target of rapamycin (Akt/mTOR) signaling pathway. METHODS: Male SD rats were ligated for 45 min and reperfused for 60 min to establish an MI/RI model. The rats were divided into sham, MI/RI model and model+ICTF 0.5, 1.0 and 2.0 mg·kg-1groups. II lead electrocardiogram (ECG) in T wave and ST segment changes were recorded. The area of myocardial infarction was determined by TTC. The protein levels and phosphorylation levels of microtubule-associated protein 2/1 light chain 3 (LC3 II/LC3 I), Beclin-1, Akt and mTOR in myocardial tissues were detected by Western blotting. The level of LC3 in myocardial tissues was detected by immunofluorescence test. RESULTS: The ECG showed that the T wave (P<0.05) and ST segment (P<0.01) of the model group were significantly higher than those of the sham group after 60 min of reperfusion, while the T wave (P<0.05) and ST segment (P<0.01) of the ICTF 1.0 mg·kg-1group were obviously lower than those of the model group. TTC staining of heart sections showed that the area of myocardial infarction in the model group was larger than in the sham group (P<0.01), while that in the ICTF 1.0 mg·kg1group was smaller than in the model group (P<0.01). Western blotting results showed that compared with the sham group, the ratios of LC3 II/LC3 I (P<0.01) and p-Beclin-1/Beclin-1 (P<0.05) in the model group were significantly increased, while Akt (P<0.01) and mTOR (P<0.05) were decreased. In addition, compared with the model group, the ratio of LC3 II/LC3 I (P<0.01) and p-Beclin-1/Beclin-1 (P<0.01) in the ICTF 1.0 mg·kg-1group was reduced, and the phosphorylation levels of Akt (P<0.01) and mTOR (P<0.01) were increased. Immunofluorescence results of frozen sections of myocardial tissues showed that LC3 protein expression increased in the model group compared with the sham group (P<0.01), but decreased in the ICTF 1.0 mg·kg-1group compared with the model group (P<0.01). CONCLUSION: ICTF has a protective effect on myocardial ischemia/reperfusion injury in rats, and its mechanism may be related to the regulation of Akt/mTOR signaling pathway to inhibit excessive autophagy.

Metabolic profiling of icaritin in rats using UHPLC-Q/TOF-MS / 中草药·英文版

Objective: To identify the in vivo metabolites of icaritin and speculate its metabolic profiling in rats. Methods: The plasma, bile, urine, and feces of rats were collected after orally administration of icaritin at a dose of 100 mg/kg and detected by an ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS/MS)in both positive and negative modes. The data of treated and control groups were compared and analyzed with the aid of Metabolynx XS software. Results: A total of 25 metabolites were identified in the biosamples, and 14 of them were reported for the first time to our knowledge. Conclusion: The main metabolite types of icaritin in rats were glucuronide conjugation, methylation, hydroxylation, reduction, and acetylation.

Reversal of multidrug resistance by icaritin in doxorubicin-resistant human osteosarcoma cells / 中国天然药物

Multidrug resistance (MDR) is one of the major obstacles in cancer chemotherapy. Our previous study has shown that icariin could reverse MDR in MG-63 doxorubicin-resistant (MG-63/DOX) cells. It is reported that icariin is usually metabolized to icariside II and icaritin. Herein, we investigated the effects of icariin, icariside II, and icaritin (ICT) on reversing MDR in MG-63/DOX cells. Among these compounds, ICT exhibited strongest effect and showed no obvious cytotoxicity effect on both MG-63 and MG-63/DOX cells ranging from 1 to 10 μmol·L. Furthermore, ICT increased accumulation of rhodamine 123 and 6-carboxyfluorescein diacetate and enhanced DOX-induced apoptosis in MG-63/DOX cells in a dose-dependent manner. Further studies demonstrated that ICT decreased the mRNA and protein levels of multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1). We also verified that blockade of STAT3 phosphorylation was involved in the reversal effect of multidrug resistance in MG-63/DOX cells. Taken together, these results indicated that ICT may be a potential candidate in chemotherapy for osteosarcoma.

Protective effect of icaritin on focal cerebral ischemic–reperfusion mice / 中草药·英文版

Objective: To investigate the protective effects of icaritin (ICT), one of the active ingredients in Epimedii Folium, on mouse model of cerebral ischemia-reperfusion (I/R) in vivo. Methods: ICR mice were subjected to an 1 h transient middle cerebral artery occlusion (MCAO) and followed by 24 h of reperfusion. Neurological deficits, infarct volume, brain edema and survive rate were measured, respectively. The levels of brain IL-1β TNF-α ROS and DNA-binding activity of NF-κB p65 were measured by ELISA kits. The levels of malondialdehyde (MDA) and activities of superoxide dismutase (SOD) were detected by spectrophotometry, and the release of nitric oxide (NO) were detected by Griess kit. Results: ICT markedly reduced the neurological deficit scores, brain edema, infarct volume and increased the survival rate of the cerebral I/R mice. The expression of IL-1β TNF-α NO, MDA and DNA-binding activity of NF-κB p65 were significantly inhibited by ICT, while the activity of SOD were up-regulated at the same time. Conclusion: ICT possessed significant neuroprotective effects in cerebral I/R mice, which might be related to prevent neuroinflammatory and oxidative damage.

Reversal of multidrug resistance by icaritin in doxorubicin-resistant human osteosarcoma cells / 中国天然药物

Multidrug resistance (MDR) is one of the major obstacles in cancer chemotherapy. Our previous study has shown that icariin could reverse MDR in MG-63 doxorubicin-resistant (MG-63/DOX) cells. It is reported that icariin is usually metabolized to icariside II and icaritin. Herein, we investigated the effects of icariin, icariside II, and icaritin (ICT) on reversing MDR in MG-63/DOX cells. Among these compounds, ICT exhibited strongest effect and showed no obvious cytotoxicity effect on both MG-63 and MG-63/DOX cells ranging from 1 to 10 μmol·L. Furthermore, ICT increased accumulation of rhodamine 123 and 6-carboxyfluorescein diacetate and enhanced DOX-induced apoptosis in MG-63/DOX cells in a dose-dependent manner. Further studies demonstrated that ICT decreased the mRNA and protein levels of multidrug resistance protein 1 (MDR1) and multidrug resistance-associated protein 1 (MRP1). We also verified that blockade of STAT3 phosphorylation was involved in the reversal effect of multidrug resistance in MG-63/DOX cells. Taken together, these results indicated that ICT may be a potential candidate in chemotherapy for osteosarcoma.

Icaritin, a Flavonoid Derived from the Herb Epimedium, Promotes Osteogenic Differentiation of MC3T3-E1 Cells

Osteoporosis is a metabolic bone disease that is characterized by low bone mass resulting from an increase in bone resorption relative to bone formation. The most current therapies for osteoporosis have focused on inhibiting bone resorption by osteoclasts. The purpose of this study is to develop new anabolic agents for treatment of osteoporosis that have fewer risks compared to conventional therapies. We searched the natural products that were derived from the traditional Asian medicines which have been used for treatment of bone related diseases. Icaritin is a flavonoid glycoside derived from the herb Epimedium which has beneficial effects on bone formation. To determine the effect of icaritin on bone formation, we examined the effect of icaritin on MC3T3-E1 cell proliferation and differentiation. For determining the effects of icaritin on proliferation, we performed the MTT assay using MC3T3-E1 cells. To evaluate whether icaritin could promote the osteogenic differentiation of MC3T3-E1 cells, alkaline phosphatase (ALP) activity and mRNA expressions of Runx2, osteocalcin (OCN), RANKL, and osteoprotegerin (OPG) were determined. Icaritin increased MC3T3-E1 cell proliferation. Icaritin increased the ALP activity of MC3T3-E1 cells on 72 hour culture in osteogenic media. mRNA expression of Runx2 was increased after 24 hour culture with icaritin. mRNA expression of osteocalcin was increased after 72 hour culture with icaritin. In addition, icaritin increased the mRNA expressions of OPG and RANKL. However, icaritin increased the mRNA expression of OPG much more than that of RANKL, and then, it increased the OPG/RANKL ratio. These results suggest that icaritin promotes osteogenic differentiation of osteoblasts and decreases osteoclast formation regulated by osteoblasts.

Protective effect of icaritin lipsome against hepatic ischemia/reperfusion injury in rats and its mechanism / 第二军医大学学报

Objective To investigate the protective effect of icaritin liposme against hepatic ischemia/reperfusion (I/R) injury in rats and its mechanisms. Methods A total of 120 male SD rats were randomly divided into four groups: icaritin liposome+I/R (ICT+I/R) group, vechicle liposome + I/R (LIP + I/R) group, I/R group and sham operation (Sham) group. Each group was randomly divided into two subgroups of 2 h and 6 h. The rats in the Sham group were only with free hilum, and the other three groups were subjected to 70% liver ischemia for 60 min. Icaritin liposome (1. 5 mg/kg) or vechile lipsome (with same volume of icaritin liposome) were intraportal venously injected in the ICT+ I/R and LIP+I/R groups at 10 min before ischemia, without any pretreatment in the I/R group. Blood and liver tissue samples in each group were obtained at 2 h and 6 h after reperfusion to measure the levels of serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST), and the contents of superoxide dismutase (SOD), malondialdehyd (MDA), nitric oxide (NO), nitric oxide synthase (NOS), inducible nitric oxide synthase (NOS), endothelial nitric oxide synthase (eNOS) and myeloperoxidase (MPO) in liver tissues. The morphology of liver tissues was observed by H-E staining. The apoptosis of liver cells and apoptosis index (AI) were calculated by TUNEL staining. Results Compared with the LIP + I/R and IR groups, ALT level, MDA and MPO contents, and AI were significantly decreased in the ICT+I/R group at 2 h after reperfusion (P<0. 05, P<0. 01). At 6 h after reperfusion, the ALT and AST levels, MDA content, and AI in the ICT+I/R group were significantly reduced, and the contents of SOD, NO, NOS, and eNOS were significantly increased compared with the LIP+I/R and IR groups (P<0. 05, P<0. 01). Conclusion Icaritin liposome can reduce liver I/R injury by increasing the contents of SOD and NO, reducing the formation of MDA, promoting the expression of eNOS, and inhibiting the accumulation of MPO and liver cell apoptosis.

Icaritin exerted effect in anti-postmenopausal osteoporosis by binding to RANKL protein target and inhibiting bone resorption / 中草药

Objective To demonstrate that icaritin (IT) inhibits bone resorption against osteoporosis by binding to RANKL protein targets. Methods The effects of RANKL and IT on the osteoporosis of ovariectomized rats were established by molecular docking technique. The effects of IT on the body weight, bone resorption serum index (ALP and TRACP-5b), bone mineral density and bone morphology of OVX rats were evaluated. Results IT could be stably docked with target protein RANKL. The body weight of IT group was significantly lower than that of OVX group (P andlt; 0.05). IT could significantly decrease the levels of serum ALP and TRACP-5b (P andlt; 0.01), and the value of femur BS/BV and Tb.Sp (P andlt; 0.01) in OVX rats. Moreover, IT significantly increased BMD, BV/TV, Tb.ThN, Tb.N values (P andlt; 0.01). Conclusion IT can inhibit osteoclast differentiation and play anti-osteoporosis by binding with RANKL protein target.

Icaritin promotes chondrogenic differentiation of BMSCs by Wnt/β-catenin signaling pathway / 中国中药杂志

To investigate the effect of icaritin (ICT) combined with GDF-5 on chondrogenic differentiation of bone marrow stromal cells (BMSCs), and discuss the action of Wnt signaling pathway, full bone marrow adherent method was used to isolate and culture SD rats BMSCs, and the cells at P3 generation were taken and divided into 6 groups： BMSCs group, ICT group, GDF-5 group, GDF-5+ICT group, GDF-5+ICT+SB216763 group, and GDF-5+ICT+ XAV-939 group. The cells were induced and cultured for 14 days. The morphology change was observed by inverted microscope. Alcian blue staining method was used to detect the changes of proteoglycans. RT-PCR was used to detect the mRNA expressions of aggrecan, Col2, Sox9, Dvl1, Gsk3β, and β-catenin. The protein expressions of collagen 2 (COL2) and β-catenin were detected by Western blot. The results indicated that, compared with the BMSCs group, gradual increase was present in proteoglycan Alcian blue staining; mRNA expressions of cartilage differentiation marker genes aggrecan, COL2, Sox9 and the protein expression of COL2, as well as mRNA and protein expressions of Wnt signaling pathway-related gene β-catenin, but with gradual decrease in Gsk3β mRNA expressions in GDF-5 group, GDF-5+ICT group and GDF-5+ICT+SB216763 group. On the contrary, compared with GDF-5+ICT group, there was a decrease in expressions of Dvl1, and β-catenin related to chondrogenic differentiation and Wnt signaling pathway, a increase in Gsk3β mRNA expression, and also a decrease in protein expressions of COL2 and β-catenin in GDF-5+ICT+XAV-939 group, with statistically significant difference between two groups. GDF-5 in combination with icaritin can induce chondrogenic differentiation of BMSCs in rats, and icaritin (ICT) can promote the chondrogenic differentiation. ICT can promote the chondrogenic differentiation of BMSCs in vitro probably by activating the Wnt/β-catenin signaling pathway.

Icaritin promotes the apoptosis of leukemia K562 cells / 白血病·淋巴瘤

Objective To investigate the effect and mechanism of the icaritin on the human cronic myeloid leukemia K562 cells. Methods The K562 cells at logarithmic growth phase were divided into the control group and the icaritin group. The cells in the control group were normally treated and the cells in the icaritin group were incubated with 8 μmol/L icaritin. Methyl thiazolyl tetrazolium (MTT) method and flow cytometry were used to examine the proliferation and apoptotic changes in the two groups after incubation for 72 h, respectively. Gene expression of p85 and Akt were detected by RT-PCR. The protein changes of p85, Akt, p-p85, p-Akt cleavage-caspase-3 and caspase-3 were detected by Western blot. Results Compared with the control group, the proliferation rate of K562 cells in the icaritin group was significantly decreased (P 0.05). Conclusion Icaritin could induce the proliferation and promote the apoptosis of K562 cell, and its mechanism may be achieved through activating the PI3K-Akt signal transduction pathway.

The effects of icaritin on the proliferation and osteogenic differentiation of rat bone marrow stromal cells / 实用口腔医学杂志

Objective:To investigate the effects of icaritin(ICT)on the proliferation and osteogenic differentiation of rat bone mar-row stromal cells(rBMSCs).Methods:rBMSCs were cultured from the bone marrow of SD rats and identified by multilineage differ-entiation assays.3,6 and 9 days after the treatment of rBMSCs of passage 4 by ICT at 1 0 -9 ,1 0 -8 ,1 0 -7 ,1 0 -6 and 1 0 -5 mol/L re-spectively,the proliferation and differentiation of the cells were examined by cck-8 and alkaline phosphatase (ALP)activity assay kit respectively.The calcium nodule formation was observed by alizarin red(AR)staining 21 days after 1 0 -9 mol/L ICT treatment. Results:Primary rBMSCs showed the typical spindle-like shape with attachment growth.rBMSCs could be induced to osteogenic and adipogenic differentiation.The proliferation of rBMSCs was inhibited but ALP activity was enhanced by ICT.1 0 -9 mol/L ICT in-cresed calcium nodule formation.Conclusion:ICT can dose-dependently inhibit the proliferation,but promote the osteogenic differ-entiation of rBMSCs.

Identification of Icaritin Metabolites in Rats by LC-MS/MS / 中草药·英文版

Objective: Icaritin is the main aglycone and also active intestinal metabolite of prenylflavonoids from the Chinese materia medica Epimedii Herba. Modern pharmacological studies have demonstrated that icaritin has a wide range of biological activities. However, its metabolites and biotransformation pathways have not yet been comprehensively investigated. The present study aims to identify icaritin metabolites in rats by using a sensitive and effective LC-MS/MS method. Methods: The plasma and urine samples of rats were collected before (blank) and after oral administration of icaritin, and subjected to liquid-liquid extraction with ethyl acetate. The full-scan LC-MS chromatograms of the plasma and urine samples were compared with those of blank samples to detect the possible metabolites, which were later detected by their product ion spectra. Results: A total of 23 metabolites were identified, and conjugated icaritins produced by glucuronidation, glycosylation, and sulfation were its major metabolites. Minor demethylation, hydrogenation, and oxidation metabolites were also found. Conclusion: Phase II metabolism is the main metabolic pathway of icaritin.

Study on Preparation and Dissolution Rate of Icaritin-Poloxamer 188 Solid Dispersion / 中国药房

OBJECTIVE:To prepare icaritin-poloxamer 188 solid dispersions in order to increase the dissolution rate of icaritin. METHODS:With poloxamer 188 as the carrier,melting method was used to prepare solid dispersions. By comparing the in vitro dissolution rates,the effects of the content of poloxamer 188(the ratios of icaritin to poloxamer 188 were 5∶1,3∶1,2∶1,1∶1,1∶3,1∶5,1∶7,1∶9,1∶11,1∶13,1∶15,1∶17,1∶19,1∶27 and 1∶31),melting temperature(60,70 and 80 ℃)and cooling tem-perature(-20,0 and 20 ℃)on the dissolution rate of icaritin in the solid dispersions were investigated,and the in vitro dissolu-tion rates of icaritin in its active pharmaceutical ingredient,physical mixture and solid dispersions were compared to confirm the for-mation of the solid dispersions. RESULTS:The dissolution rate of icaritin in the prepared solid dispersions increased to some extent as the proportion of the carrier increased. When the ratio of icaritin to the carrier was 1∶17-1∶27,the dissolution rate of icaritin at 120 min was above 90%. Where melting temperature and cooling temperature were respectively determined as 60 ℃ and 0 ℃ after comprehensive comparison,the dissolution rate of icaritin in the solid dispersions was 1.5 times as much as that in the physical mix-ture at 30 min. CONCLUSIONS:The prepared solid dispersion has a significantly higher dissolution rate of icaritin.