Identification of chemical constituents in Zhachong Shisanwei Pills by UPLC-Q-TOF-MS/MS / 中国中药杂志

Zhachong Shisanwei Pills, composed of 13 Chinese medicinal materials, are used for treating the diseases such as hemiplegia, pain of muscles and bones, rheumatism, and joint pain. The chemical composition and pharmacodynamics of Zhachong Shisanwei Pills have not been reported. Ultra-performance liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS/MS) was employed to quickly identify the chemical components of Zhachong Shisanwei Pills, which was performed with Shim-pack GIST C_(18) column(4.6 mm×150 mm, 5 μm). The gradient elution was conducted with methanol-0.05% acetic acid as the mobile phase. Electrospray ionization mass spectrometry(ESI-MS) was carried out in both positive and negative ion modes. The compounds were identidied based on accurate relative molecular weight, fragment ion species, and the MS data of reference substances and in literature. In conclusion, a total of 98 compounds were identified, including 19 organic acids, 36 flavonoids, 13 volatile oils, 8 tannins, 5 2-(2-phenylethyl)chromones, 5 amino acids, 3 sesquiterpenoids, 3 alkaloids, and 2 other compounds. This study characte-rized the chemical components of Zhachong Shisanwei Pills rapidly for the first time, laying a foundation for further research on the pharmacodynamic material basis and quality evaluation.

Effects of verapamil on pharmacokinetics of Y101 in vivo in rats / 中国药理学通报

Aim To develop a liquid chromatography electrospray-ionization tandem mass spectrometry (LC- MS/MS) method for simultaneous determination of bentysrepinine (Y101) and its metabolites M8 and M9 in rat plasma and to investigate the effect of verapamil, an inhibitor of P-glycoprotein (P-gp) , on the pharma¬cokinetics of Y101, a substrate of P-gp, in rats. Methods SD rats were divided randomly into two groups; ( 1) Y101 only as a control group, received an oral dose of 60 mg • kg"1 Y101; (2) Verapamil plus Y101 as an experimental group, received an oral dose of 60 mg • kg"1 Y101 in combination of 25 mg • kg"1 verapamil. The plasma concentrations of Y101 and its metabolites were determined by LC-MS/MS method af¬ter intragastric administration, and the pharmacokinetic parameters were calculated using non-compartmental a- nalysis. Results We successfully developed and fully validated a LC-MS/MS method, which simultaneously determined the concentration of Y101 and its metabo¬lites in rat plasma. The AUC0_t for Y101 and M9 in experimental group increased to 1.71-fold and 1.58- fold in comparison of control group. At the same time, the plasma clearance of Y101 and M9 decreased to 60% of control. However, we did not find any differ¬ence in AUC0_l and plasma clearance for M8 between two groups. Conclusions The validated LC-MS/MS method is sensitive and rapid for the determination of Y101 and its metabolites in rat plasma and was suc¬cessfully applied to the pharmacokinetic study in rats. Verapamil, a P-gp inhibitor, significantly increases the exposure of Y101 and its metabolites in vivo, indicating the adjustment of Y101 dosage for combined adminis¬tration is needed in clinical practice.

Investigation of the metabolites of five major constituents from Berberis amurensis in normal and pseudo germ-free rats / 中国天然药物

Berberis amurensis (Berberidaceae) is a traditional Chinese medicine, which is often used to treat hypertension, inflammation, dysentery and enteritis. It contains alkaloids, mainly including berberine, berbamine, magnoflorine, jatrorrhizine and palmatine. Berberis amurensis extracts (BAEs) is often orally taken. Oral herbs might be metabolized by intestinal bacteria in the small intestine. However, the interaction between the herb and the gut microbiota is still unknown. In the current study, UPLC/Q-TOF-MS/MS combined with Metabolitepilot and Peakview software was used to identify the metabolites of BAEs in anti-biotic cocktail induced pseudo germ-free rats and normal rats. As a result, a total of 46 metabolites in normal rats were detected and its main metabolic pathways include demethylation, dehydrogenation, methylation, hydroxylation, sulfation and glucuronidation. Only 29 metabolites existed in pseudo germ-free rats. Dehydrogenated metabolites (M29, M30, M34 and M36), methylated metabolites (M33, M41 and M46) and other metabolites were not detected in pseudo germ-free rats. The result implied that the intestinal bacteria have an influence on the metabolism of BAEs. Furthermore, this investigation might contribute to the understanding of the metabolism of BAEs, and further promote its clinical application.

In vitro metabolism of liquiritigenin in liver microsomes of different species / 中草药

Objective: To compare in vitro metabolic differences of liquiritigenin, an aglycone of liquiritin, among liver microsomes of different species, which would provide reference for further research and development of liquiritin. Methods: Metabolic stability and metabolic biotransformation were investigated after liquiritigenin was incubated with rat, mouse, human, dog, and monkey liver microsomes. Metabolic stability was evaluated using a substrate depletion approach, and the results were reported as "% liquiritigenin remaining", which was then used to calculate the in vitro half-life (t1/2). Metabolic biotransformation was characterized by metabolite profiling. Results: In liver microsomal incubation systems of five species, the t1/2 values of liquiritigenin for phase I were as follows: rat < mouse < human < monkey < dog, whereas for phase II, the metabolic rates were all fast and the remaining of liquiritigenin were all below 50% in 5 min except mouse. In addition, phase II metabolite profiling in monkey liver microsomes was identical to that of human, but marked differences were found between other species and human. Conclusion: The metabolic characteristics of liquiritigenin in monkey liver microsomes is most similar to that of human, then followed by dog, and marked differences existed between rat, mouse and human. Therefore, monkey or dog could be the animal model for further preclinical pharmacokinetic and toxicological studies.

In vivo metabolic pathway of naringenin in rats / 中草药

Objective: To study the in vivo metabolic pathway of naringenin in rats, which would provide reference for its further research and development. Methods: A liquid chromatography with hybrid quadruple-linear ion trap mass spectrometer (LC-QTRAP-MS) was applied to identify the metabolites of naringenin in bile, urine, feces and plasma after ig administration to rats. The fragmentation patterns of naringenin were also used to analyze possible metabolic pathways in the study. Results: There were altogether 14 metabolites detected. The possible metabolic pathways of naringenin were oxidation, methylation, glucuronidation and sulfation in rats. Conclusion: In vivo metabolism studies show that naringenin undergoes extensive metabolism, including phase I and phase II biotransformation, and the phase II glucuronide and sulfate conjugates are primary metabolites. The relative quantitative amounts of metabolites excreted from urine are almost equivalent to that of the parent naringenin, while a large majority of metabolites are conjugated ones in bile, which are subsequently transformed to the parent in intestinal tract and appeared in feces.

The in vitro inhibition and induction of cytochrome P450 activities by bentysrepinine: a novel candidate of anti-hepatitis B virus drug / 药学学报

Bentysrepinine (Y101), a derivative of phenyalanine dipeptide, has a novel mechanism in the treatment of hepatitis B virus (HBV) infection with a good anti-HBV effect. In the present study, a fluorometric-based high throughput method using cytochrome P450 (CYP) screening kit was adopted to evaluate in vitro inhibition potential of Y101 on CYP isoenzymes by calculating remaining enzyme activities and inhibitory potential (IC50 values) using the determined values of fluorescence intensity. The result showed that Y101 exhibited little activity in the inhibition of CYP1A2, CYP3A4, CYP2C9, CYP2C19 and CYP2D6 (IC50 > 100 μmol·L-1). Y101 was used to treat human primary hepotocytes for 72 h, and the enzyme activities of CYP1A2, CYP2B6 and CYP3A4 were determined with a cocktail of probe substrates for the three CYP isoforms. The metabolites were simultaneously determined using a LC-MS/MS method. Y101 had no activity in the induction of CYP1A2, CYP2B6 and CYP3A4 on the basis of the following results:① The ratio of enzyme activities between test and control groups were all below than 1 (varied from 0.662 to 0.928); ② The induction potential of Y101 were lower than forty percent compared with that of positive groups. The above results suggest that Y101 has little activity in the regulation of metabolic drug-drug interactions based on the CYP isoform changes following co-administration of drugs.

The in vitro transport mechanism of bentysrepinine:a novel anti-hepatitis B virus drug candidate / 药学学报

Bentysrepinine (Y101), a derivative of phenylalanine dipeptide, is a novel drug candidate for the treatment of hepatitis B virus (HBV) infection. Our previous preclinical pharmacokinetic study showed that its in vivo absorption and distribution characteristics were probably related to transmembrane transport after Y101 was administered intragastically in rats. In this study, Caco-2 and MDCK-MDR1 cell models were used to investigate interactions between Y101 and P-gp through the apparent permeation coefficient (Papp) and efflux ratio (RE); the results showed that Y101 was a substrate of P-gp. In addition, gene-transfected cell models, HEK293-hOATP1B1, HEK293-hOATP2B1 and CHO-PEPT1 were used to evaluate the affinity to OATP1B1,

Determination of tamsulosin in dog plasma by a high sensitive liquid chromatography-tandem mass spectrometric method / 药学学报

To develop and validate a liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of tamsulosin in dog plasma after oral administration of controlled-release tablet of tamsulosin hydrochloride, the samples and the internal standard, diphenhydramine, were extracted from dog plasma by n-hexane-dichloromethane (2 : 1), and separated on a Bonchrom XBP-C18 column using a mobile phase consisted of methanol-acetonitrile-ammonium formate (10 mmol x L(-1)) (30 : 40 : 30, v/v/v), at a flow rate of 0.4 mL x min(-1). Mass spectrometric detection was operated on a triple quadrupole tandem mass spectrometer equipped with atmospheric pressure chemical ionization (APCI) source in positive mode. Quantification was performed using selected reaction monitoring (SRM) of the transitions m/z 409 --> 228 for tamsulosin and m/z 256 --> 167 for the internal standard, respectively. The linear concentration ranges of the calibration curves for tamsulosin were 0.02 - 50 ng x mL(-1). The lower limit of quantification was 0.02 ng x mL(-1). The accuracy ranged from -2.61% to 8.82% in terms of relative error (RE). The intra- and inter-day relative standard deviation (RSD) across three-run validations were lower than 9.72%. The method was proved to be highly sensitive, selective, and had been successfully applied to the pharmacokinetic study after an oral administration of 0.4 mg tamsulosin hydrochloride controlled release preparations to dogs.