An on-line solid phase extraction-high performance liquid chromatography ( SPE-HPLC ) system was applied in the plasma pharmacokinetic study of highly active anti-cancer compound tyrosine kinase inhibitors (TEB-415) in mouse. The on-line SPE-HPLC method associated with Ultimate3000 system which was applied to the determination of the blood drug level of TEB-415 in mouse plasma. C18 column ( Venusil MP, 150 mm × 4. 6 mm, 5μm) was used as analytical column and the mobile phase consisted of acetonitrile-5 mmol/L monopotassium phosphate buffer ( pH 3 . 5 ) at a flow rate of 1 . 0 mL/min was used as the isocratic elution. An MF Ph-1 column (10 mm×4 mm, 5 μm) was used as on-line SPE column, and water and water-acetonitrile were used as the washing solvent and elution solvent respectively. The detection wavelength was set at 262 nm. The pharmacokinetic parameters were calculated by WinNonlin 5. 2 software. The linear range of the calibration curve was between 100 and 20000 μg/L, and the limit of qualification was 20 μg/L. The extraction recovery was between 90 . 5% and 94 . 6%. The RSD of intra-day and inter-day precision was less than 3. 5%. The accuracy of short-term stability, freeze-thaw stability and long-term stability were between 91. 49% and 101. 96%. After oral medication, the mean peak time (Tmax) of TEB-415 in mice was 5. 29 h, and the mean maximum concentration ( Cmax) was 3403μg/L. The area under the curve ( AUC) of TEB-415 was 24600 μg/L·h. This drug's mean half-life was 3. 84 h, and its mean retention time (MRT) was 6. 56 h. These parameters suggested that TEB-415 had appropriate rate of absorption and elimination with preferable bioavailability.

Traditional Chinese ancient prescriptions have been used for treatment of liver cancer for a long history and the scientific and rational compatibility is a great wealth for modern research and development (R&D) of new drugs. The research and development of new drugs are often accompanied with a large investment, a long cycle and a high risk, especially for the anti-tumor drugs R&D which are facing more risks and lower successful rate. In this research, the regularity of compatibility of drugs was analyzed from 124 anti-hepatoma ancient prescriptions by computer program. The results can offer help to the R&D of anti-hepatoma new drugs and reduce the risk of drug screening. In addition, we surveyed 22 companies in this field from six provinces such as Beijing, Shanghai, Tianjin and so on and obtained 240 risk assessment questionaires. Then we used qualitative analysis method to interpret the greatest impacts for the risks in the process of R&D, production and sales of anti-hepatoma new drugs. The study provides a basis for anti-liver cancer drugs R&D researchers, who can take effective measures to reduce the R&D risks and improve successful rate.
Carcinoma, Hepatocellular ; drug therapy ; history ; China ; Drug Discovery ; history ; Drug Incompatibility ; Drug Prescriptions ; history ; Drugs, Chinese Herbal ; history ; therapeutic use ; History, Ancient ; Humans ; Liver Neoplasms ; drug therapy ; history ; Research ; history

OBJECTIVETo develop an HPLC method for determination of the four effective components (genistin; rutin; quercetin; genistein) in Huaijiao pill.

METHODThe chromatographic separation was performed on a Shim-packODS (4.6 mm x 150 mm, 5.0 microm) eluted with a mobile phase of MeOH-H2O-HAc (40:60:0.25). The detection wavelength was set at 256 nm and column temperature was set at 30 degrees C .

RESULTNice linear relation between the peak area and injected amount exists when the amount is within 0.059-0.352 microg for genistin, within 0.435-2.610 microg for rutin, within 0.020-0.121 microg for quercetin and within 0.053-0.319 microg for genistein. The correlation coefficient of each component is 0.999 6, 0.998 2, 0.998 9 and 0.999 9 respectively. The average recoveries of the four components are from 98.7% to 100.2%. The RSD of each group are 1.21%, 1.36%, 0.47% and 1.54% (n = 5).

CONCLUSIONThe method was accurate, repeatable and suitable to determine four effective components in Huaijiao pill. It can be use for quality control of Huaijiao pill.

Chromatography, High Pressure Liquid ; methods ; Drug Combinations ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Genistein ; analysis ; Isoflavones ; analysis ; Plants, Medicinal ; chemistry ; Quercetin ; analysis ; Reproducibility of Results ; Rutin ; analysis ; Sophora ; chemistry

BACKGROUNDThe development of regenerative therapies using derivatives of embryonic stem (ES) cells would be facilitated by a non-invasive method to monitor transplanted cells in vivo, for example, magnetic resonance imaging of cells labeled with superparamagnetic iron oxide (SPIO) nanoparticles. Although ES cells have been labeled with SPIO particles, the potential adverse effects of the label have not been fully examined. The objective of this study was to determine whether SPIO labeling affects murine ES cell viability, proliferation, or ability to differentiate into functional endothelial cells (ECs).

METHODSCross-linked iron oxide (CLIO, an SPIO) was conjugated with human immunodeficiency virus transactivator of transcription (HIV-Tat) peptides, and murine ES cells were labeled with either CLIO-Tat, CLIO, or HIV-Tat. After labeling, ES cells were cultured for 4 days and Flk-1(+) ES cells identified and sorted by immunocytochemistry and fluorescence activated cell sorting (FACS). Flk-1(+) cells were replated on fibronectin-coated dishes, and ECs were obtained by culturing these for 4 weeks in endothelial cell growth medium supplemented with vascular endothelial growth factor (VEGF). ES cell viability was determined using trypan blue exclusion, and the proportion of SPIO(+) cells was evaluated using Prussian blue staining and transmission electron microscopy. After differentiation, the behavior and phenotype of ECs were analyzed by reverse transcription-polymerase chain reaction, flow cytometry, immunocytochemistry, DiI-labeled acetylated low-density lipoprotein (AcLDL) uptake, and Matrigel tube formation assay.

RESULTSCLIO-Tat was a highly effective label for ES cells, with > 96% of cells incorporating the particles, and it did not alter the viability of the labeled cells. ECs derived from CLIO-Tat(+) ES cells were very similar to murine aortic ECs in their morphology, expression of endothelial cell markers, ability to form vascular-like channels, and scavenging of AcLDL from the culture medium.

CONCLUSIONSCLIO-Tat is a highly effective label for ES cells and does not adversely affect cell viability, differentiation, or behavior. CLIO-Tat could be a useful marker for the non-invasive monitoring of transplanted stem cells.

Animals ; Cell Differentiation ; drug effects ; Cell Line ; Cell Survival ; drug effects ; Embryonic Stem Cells ; cytology ; drug effects ; Endothelial Cells ; cytology ; drug effects ; Ferric Compounds ; chemistry ; Flow Cytometry ; Immunohistochemistry ; Mice ; Microscopy, Electron, Transmission ; Nanoparticles ; adverse effects ; chemistry ; Reverse Transcriptase Polymerase Chain Reaction

Apolipoprotein AI (apo AI), the major protein component of human high-density lipoprotein (HDL), is a single-chain polypeptide of 243 amino acids. Several epidemiological studies have shown that the plasma concentrations of HDL has the role of reverse cholesterol transport (RCT) and inversely correlated with the incidence of coronary artery disease. Because apo AI lacks post-translational modifications, it is convenient to express human apo AI in Escherichia coli expression system. However, there is a poor stability of the mRNA and the apo AI protein in E. coli, it is difficult to express mature apo AI in recombinant bacteria, moreover, even as a fusion protein, apo AI is still sensitive to degradation and can not be cleaved efficiently from the fusion tags. In contrast, proapolipoprotein AI (proapo AI, having an additional polypeptide containing the amino acids Arg-His-Phe-Trp-Gln-Gln at the amino-teminal of the mature protein) proved stable and undegraded in Escherichia coli, and therefore, in this research, an expression system of E. coli including a plasmid of P(R)P(L) tandem promoter was adapted to produce proapo AI. Furthermore, site-directed mutagenesis of the proapo AI cDNA was performed to generate a Clu8Asp mutation in the amino-terminal sequence of proapo AI which created an acid labile Asp-Pro peptide bond between amino acid 8 and 9, and permitted specific chemical cleavage to remove pro-peptide. After inducing with a shift of temperature, yields of recombinant proapo AI achieved about 40% of total cell protein and the recombinant proapo AI expressed proved as a form of inclusion body in cells, so protein need to renature. First of all, the protein was dissolved in buffer with denaturant, and renaturation was carried out on a hydrophobic interaction column (Phenyl Sepharose), ion-exchange chromatography and gel-filtration chromatography were then used to further purify the protein. The purified recombinant apo AI was detected by a set of tests including Western-blotting, Circular dichroism spectra and lipid-binding test, the results shown that recombinant apo AI has similar structural and lipid-binding properties identical to those of native plasma apo AI, which facilitates further research and application.
Apolipoprotein A-I ; biosynthesis ; genetics ; Chromatography, Ion Exchange ; methods ; Escherichia coli ; genetics ; metabolism ; Humans ; Mutagenesis, Site-Directed ; Mutation ; Protein Precursors ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

The type 2 diabetes rat model was induced with high-sugar and high-fat diet combined with low-dose STZ. This study investigated the hypolipidemic mechanism of Coptis chinensis and C. deltoidea. After 30 days of administration, HOMA-IR and the content of TG in serum were detected, and the expressions of SCAP, SREBP-1c were tested by the method of Western blot and Real-time PCR analysis. The test results showed that both components can significantly alleviate insulin resistance and down-regulate the expressions of SREBP-1c and SCAP in liver tissue of type two diabetes mellitus. Compared with the control group, there were significant differences in relevant protein expression (<0.05, <0.01). This indicates that the inhibition of SREBP-1c and SCAP expressions may be the hypolipidemic mechanism of Coptidis Rhizoma on type 2 diabetes mellitus. The results also showed that C. deltoidea has a better efficacy in lipid elimination, but a weaker hypoglycemic effect against C. chinensis.

Objective:To investigate the protective effect of ferulic acid on PC12 cells injured by H₂O₂ and the molecular mechanisms. Method:The oxidative stress model was established by treating PC12 cells with H₂O₂, and then different dosages of ferulic acid (1, 10, 100 μmol·L^-1) were used for intervention. Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the cell viability,lactate dehydrogenase (LDH) and malondialdelyde (MDA) in cell supernatant, and superoxidedismutase (SOD) in cells was tested by biochemical method respectively. Insulin-like growth factors-1 (IGF-1) mRNA and protein expressions were detected by Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) and Western blot. Result:The 24 h intervention with different dosages of ferulic acid (1, 10, 100 μmol·L^-1) could significantly improve the oxidative damage of PC12 cells induced by H₂O₂, compared with the model group, ferulic acid at 1,10,100 μmol·L^-1 significantly increased PC12 cells viability,significantly decreased LDH and MDA content in cell supernatant (P<0.05, P<0.01), and enhanced SOD activity(P<0.01). Real-time PCR and Western blot showed that compared with control group,IGF-1 mRNA and protein expressions in model group decreased significantly (P<0.01), compared with model group, IGF-1 mRNA and protein expressions in ferulic acid group increased significantly (P<0.01). Conclusion:Ferulic acid exerts a protective effect on H₂O₂-inducing PC12 cells injury,which might be related to insulin signaling pathways.

Fructus Arctii is a traditional Chinese medicine. The main counterfeit species are the seeds of Arctium tomentosum, Onopordum acanthium, Silybum marianum, Saussurea costus, Amorpha fruticosa. Traditional identification methods or molecular barcoding techniques can identify Fructus Arctii and its counterfeit species. However, the identification of the mixture of it and its spurious species is rarely reported. In this paper, we sequenced the ITS2 sequences of Fructus Arctii and 5 kinds of spurious species mix powder by high-throughput sequencing to identify the mixed powder species and providing new ideas for the identification of Fructus Arctii mix powder. The total DNA in mixed powder was extracted, and the ITS2 sequences in total DNA was amplified. Paired-end sequencing was performed on the DNA fragment of the community using the Illumina MiSeq platform. The sequence was analyzed by the software FLASH, QIIME and GraPhlAn etc. The results showed that the high quality ITS2 sequences of 39910 mix samples were obtained from the mixed samples, of which the total ITS2 sequence of the samples genus was 34 935. Phylogenetic analysis showed that the samples contained Fructus Arctii, A. tomentosum, O. acanthium, S. marianum, S. costus and A. fruticosa. Using ITS2 sequences as DNA barcodes, high-throughput sequencing technology can be used to detect the Fructus Arctii and its spurious specie in mixed powder, which can provide reference for the quality control, safe use of medicinal materials of Fructus Arctii and the identification of mixed powder of traditional Chinese medicine.
Arctium ; chemistry ; classification ; DNA Barcoding, Taxonomic ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drug Contamination ; Drugs, Chinese Herbal ; standards ; Fabaceae ; Fruit ; High-Throughput Nucleotide Sequencing ; Milk Thistle ; Onopordum ; Phylogeny ; Saussurea

Objective:To investigate the protective effect and molecular mechanism of Angelicae Sinensis Radix-Chuanxiong Rhizoma medicated serum （ASRCRS） against oxidative damage of PC12 cells induced by H₂O₂. Method:Oxidative damage of PC12 cells was induced by H₂O₂ in vitro， and intervention was performed in the low-， medium-， and high-dose ASRCRS groups with a final volume fraction of 15%. The cell viability was determined by methyl thiazolyl tetrazolium （MTT） assay. Cell morphology was observed by an inverted fluorescence microscope. The content of lactate dehydrogenase （LDH） and malondialdehyde （MDA）， the activity of superoxide dismutase （SOD）， and the distribution of reactive oxygen species （ROS） in the cell supernatant were detected by the kits. Cell apoptosis was detected by Annexin V-FITC/PI double staining. The protein expression levels of nuclear factor E₂-related factor 2 （Nrf2）， Kelch-like epichlorohydrin associated protein-1 （Keap1）， heme oxygenase-1 （HO-1）， and SOD1 were detected by Western blot. Result:Oxidative damage was induced by 300 μmol·L^-1 H₂O₂ for 24 hours. Compared with the normal group， the model group showed abnormal cell morphology， reduced cell viability （P<0.01）， increased LDH and MDA （P<0.01）， blunted SOD activity， elevated intracellular distribution of ROS， down-regulated protein expression of Nrf2， HO-1， and SOD1 （P<0.05， P<0.05）， and up-regulated protein expression of Keap1 （P<0.01）. Compared with the model group， ASRCRS groups displayed improved cell morphology， increased cell viability， inhibited cell apoptosis， potentiated SOD activity （P<0.01）， suppressed release of LDH （P<0.01） and generation of ROS， decreased content of MDA （P<0.01）， up-regulated protein expression of Nrf2， HO-1 and SOD1 （P<0.05， P<0.01）， and down-regulated protein expression of Keap1 （P<0.01）. Conclusion:ASRCRS could protect PC12 cells from oxidative damage induced by H₂O₂ by up-regulating the expression of Nrf2 to activate the Nrf2/antioxidant response element （ARE） signaling pathway， enhancing the ability to resist oxidative damage， and inhibiting cell apoptosis.

The patents of acupuncture and moxibustion in China and abroad was analyzed, aiming to provide support for the innovative development of acupuncture industry. With the China Think Tank of Patent of Traditional Chinese Medicine and the PatSnap database as data sources, based on the mathematical statistics method, the application trend, legal status, patent types, transformation and distribution of major technical fields of acupuncture patents in China and abroad were analyzed. As a result, a total of 53,422 acupuncture patents were screened, involving 49 countries and 4 organizations. The patent types were mainly utility model patents. Although the application number of acupuncture patent had increased rapidly, the average patent conversion rate was generally low, approximately 4%. In the context of global economic integration, the acupuncture industry is developing at a high speed. It is suggested to take advantage of the "Belt and Road Initiative" to improve the international acceptance of acupuncture and moxibustion, adhere to the principle of attaching equal importance to the number and quality of patents, promote the in-depth cooperation of industry-university-research, and promote high-quality development of acupuncture and moxibustion.
Humans ; Moxibustion ; Acupuncture Therapy ; China ; Medicine, Chinese Traditional ; Databases, Factual