Determination of prucalopride in human plasma by LC-MS/MS analysis / 中国药理学通报

Aim To develop an LC-MS/MS method for the determination of prucalopride(PCP)in human plasma.Methods Prucalopride -13CD3(dPCP)was used as the internal standard.The analytes were extracted from human plasma through liquid-liquid extraction method using ethyl acetate, followed by being dried, and then the reconstitution was injected into LC-MS/MS systems.Agilent ZORBAX SB C18(3.0×100 mm, 3.5 μm)column and isocratic elution system composing of methanol and 1 mmol·L-1 ammonium acetate(80:20, V/V)provided chromatographic separation of PCP and dPCP.AB Sciex API4000 mass spectrometer equipped with an electrospray ionization source in positive ion mode was employed for mass detection, and data acquisition was carried out in multiple reaction monitoring(MRM)mode.The mass transition ion-pair was followed as m/z 368.4/196.0 for PCP and m/z 374.4/198.0 for dPCP.Results PCP and dPCP were eluted at 3.6 min, with no interference in human blank plasma.PCP in human plasma showed good linearity over the concentration range of 0.058 96-7.547 μg·L-1 with the correlation coefficient of 0.996 3-0.999 6.The lower limit of quantitation of this method was 0.058 96 μg·L-1.The intra-batch and inter-batch accuracy ranged from 98.29% to 108.2%, with good precision(CV<5.2%).The average matrix factors of normal, haemolysed and lipaemic matrix human samples all ranged from 96.48% to 106.3% with CV less than 8.39%.The average extraction recoveries of PCP at low, medium and high concentrations were 89.88%, 95.27% and 94.52% respectively, with CV less than 7.21%.PCP was stable in human samples after 6 h at room temperature, 60 h at -20 ℃, 56 days or three freeze-thaw cycles at -80 ℃; meanwhile, the processed plasma samples remained stable after being stored for 24 hours in autosampler at 8 ℃.Furthermore, PCP in human blood samples was proved to be stable after 4 h at room temperature.Conclusions The present LC-MS/MS method for the determination of PCP in human plasma was convenient, accurate, sensitive, stable, specific and reproducible and was proved to be suitable for the clinical pharmacokinetics and bioequivalence studies of PCP preparations.

Investigation of Pharmacodynamic Material Basis and Mechanism of Qianyang Yuyin Granules Against Hypertensive Renal Damage Based on LC/Q-TOF-MS and Network Pharmacology / 中国实验方剂学杂志

Objective:To systematically study the chemical components of Qianyang Yuyin granules and explore its main pharmacodynamic substances and mechanism in the prevention and treatment of hypertensive renal damage. Method:Liquid chromatography coupled with quadrupole time-of-flight mass spectrometry （LC/Q-TOF-MS） was employed to comprehensively analyze the chemical components of Qianyang Yuyin granules. Agilent Poroshell 120 SB-C₁₈ column （3.0 mm×100 mm， 2.7 μm） was used， flow rate was 0.4 mL·min^-1， electrospray ionization （ESI） was applied and operated in positive and negative ion modes， the acquisition range was <italic>m</italic>/<italic>z</italic> 25-1 000. Mobile phase in positive ion mode consisted of water+10 mmol·L^-1 ammonium formate+0.125% formic acid+0.1% methanol （A）-［acetonitrile-water （9∶1）+10 mmol·L^-1 ammonium formate+0.125% formic acid］ （B）， and in negative ion mode consisted of water+10 mmol·L^-1 ammonium formate+0.1% methanol （A）-［acetonitrile-water （9∶1）+10 mmol·L^-1 ammonium formate］ （B） with the gradient elution （0-3.5 min， 5%B； 3.5-4 min， 5%-10%B； 4-9 min， 10%-25%B； 9-18 min， 25%-30%B； 18-25 min， 30%-50%B； 25-27 min， 50%-90%B； 27-32 min， 90%B； 32-33 min， 90%-5%B； 33-39 min， 5%B）. According to the information of the accurate mass， the multistage fragment ions， the mass spectrometric data of the standard substances and the relative reference literature， the structures of the chemical components in Qianyang Yuyin granules were identified. Based on the identified components， network pharmacology study， including target prediction and functional enrichment was applied to screen out the main active substances against hypertensive renal damage， and explore the potential mechanism. Result:A total of 99 chemical components were identified， from which 43 active substances and 48 key targets were screened out. The key components contained kaempferol， quercetin， ferulic acid， luteolin， caffeic acid methyl ester， cinnamic acid， aloe-emodin， emodin， gallic acid， <italic>N</italic>-<italic>trans</italic>-feruloyltyramine， isoorientin， 8-<italic>O</italic>-feruloylharpagide， ethyl caffeate， isookanin， cyasterone， 2，3，5，4'-tetrahydroxystilbene-2-<italic>O</italic>-<italic>β</italic>-<italic>D</italic>-glucopyranoside， loganin， alisol B-23-acetate and harpagide. The key targets included vascular endothelial growth factor A （VEGFA）， serine/threonine protein kinase 1 （AKT1）， Jun proto-oncogene （JUN）， etc. Conclusion:Qianyang Yuyin granules mainly exert the effects of removing heat from the liver， tonifying the kidney and removing blood stasis via modulation of vascular endothelium， angiogenesis， inflammatory reaction， oxidative stress， immune response and so on.

Dauricine inhibiting the cell proliferation and inducing the cell apoptosis of human pancreatic cancer cells line SW1900 / 解剖学报

Objective To discuss the proliferation inhibition and apoptosis induction of human pancreatic cancer cell line SW1900 by dauricine and its possible mechanism. Methods The MTT colorimetric method was used to detect the inhibitory effects of cell viability. The apoptosis rate was tested by the Annexin Ⅴ-FITC / PI fluorescent staining of flow cytometric method . The expressions of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and B-cell lymphoma-2 (Bcl-2) were detected by Real-time PCR and Western blotting assay. Results MTT assay showed that dauricine significantly inhibited the proliferation of SW1900 cells and the inhibitory effect was enhanced with the increasing of dauricine concentration, F = 783. 7, P < 0. 001. The apoptosis of 3 groups cells were (4. 34 ± 1. 30) % (0 mg / L dauricine), (14. 94±1. 94) % (6 mg / L dauricine) and (22. 68±3. 61) % (12 mg / L dauricine) . The mean difference was statistically significant among the three groups (F = 58. 52, P < 0. 001) . Dauricine could significantly induce apoptosis human pancreatic cancer cells with dose-dependent manner. Real-time PCR showed that the gene expressions of PI3K, Akt and Bcl-2 were lower obviously (PI3K mRNA, F = 101, P = 0. 01; Akt mRNA, F = 1666, P < 0. 01; Bcl-2 mRNA, F = 753, P<0. 001) with dose-dependent manner. Western blotting assay also showed that the protein expression of PI3K, Akt and Bcl-2 was down-regulated with dose-dependent manner. Conclusion Dauricine has proliferation inhibition and apoptosis inducement effect on human pancreatic cancer cells line SW1900. This function may be concerned with the regulation of PI3K / Akt signal pathway and lower Bcl-2 expression.

Screen astragalosides from Huangqi injections by LC-TOF-MS-based mass defect filtering approach / 中国中药杂志

The samples of Huangqi injection (HI) were analyzed by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-TOF-MS), and both positive and negative ion modes were employed to obtain the LC-TOF-MS analysis information of chemical compounds in HI. Then the mass defect filtering (MDF) approach, which was developed based on the previously published articles, was utilized to rapidly screen the astragalosides from the obtained LC-TOF-MS data. Each screened astragaloside was confirmed by the presence of no less than 2 quasi-molecular ions. All the screened astragalosides were then tentatively assigned according to the parent ion and daughter ion information. Finally, a total of 62 astragalosides were screened and characterized from the HI samples, including 15 new detected ones. The identification results indicated that acetylation, hydrogenation, dehydrogenation, methoxylation and hydration might be the major conversion reactions involved in the formation of the astragalosides. The LC-TOF-MS-based MDF approach was proved to be a feasible and efficient tool to screen the chemical constituents in complex matrices such as herbal medicines.

Study on biomarker of Tripterygium wilfordii in treatment of rheumatoid arthritis based on PK/PD / 中国中药杂志

To observe the serum samples and the anti-inflammatory effect of Tripterygium wilfordii in treating RA by using the pharmacokinetic-pharmacodynamic model, make a correlation analysis on concentration-time and effect-time curves, and explore RORγt, IL-17, STAT3, IL-6 mRNA transcriptional levels in rats by PCR. Methotrexate, tripterine and high-dose T. wilfordii could down-regulate RORγt, IL-17, STAT3, IL-6 mRNA transcriptional levels in AA rat lymph nodes. The study on PK-PD model showed correlations between inflammatory factors and blood concentration of T. wilfordii. T. wilfordii and its main active constituent tripterine could show the inflammatory effect and treat RA by inhibiting IL-17 cytokine.