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@#A new method based on rat liver microsomes and chromatographic fingerprint comparison was established to investigate the possible herb-drug interactions between Liuwei Dihuang Pills(LDP)and nifedipine(NIF). LDP and NIF were incubated simultaneously with rat liver microsomes at 37 °C for 60 min in a shaking water bath. The separation was achieved on a C18 column using 0. 1% formic acid solution and acetonitrile as mobile phase with a liner gradient program. The flow rate was set at 1. 0 mL/min. Detection was achieved by UV light at 240 nm. To evaluate the interactions between LDP and NIF, the similarity of the fingerprinting chromatograms before and after co-incubation was calculated by similarity evaluation system for chromatographic fingerprint of TCM. Furthermore, pharmacokinetic experiments in rat suggested that there was no significant difference in the pharmacokinetic parameters of the typical components in LDP.
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OBJECTIVE: To investigate the hepatic toxicity of 8 monomers in Polygonum multiflorum using a combination of UDPglucuronic acid transferase 1A1(UGT1A1 enzyme). METHODS: Bilirubin was used as the substrate for UGT1A1. Incubation method in RLM in vitro was adopted to test the apparent inhibition constants(Ki) of different components. Furthermore the structure-activity relationship between the 8 components and UGT1A1 was analyzed. RESULTS: The inhibition effects on UGT1A1 enzyme of the 8 components were in the following sequence: emodin-8-O-glc > emodin > citreorosein > (+) -catechin > gallic acid > physcion > rhein > emodin-6-O-glc. Moreover, there was a structure-activity relationship, and it was presumed that the 6-position hydroxyl group is an active and necessary group. CONCLUSION: The established method in vitro is stable and feasible. Experimental results shows that the enzyme inhibition has structural selectivity, which provides an experimental basis for predicting the enzyme inhibition activity of the analogues of components of Polygonum multiflorum.
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OBJECTIVE: To identify the specific cytochrome P450 (CYP) enzymes involved in the metabolism of dipfluzine hydrochloride (Dip) in the rat liver microsomes. METHODS: The rat liver microsomes were prepared and incubated with Dip. The Dip metabolites (M1, M2, M4 and M5) were identified by LC-MS/MS, and the CYP isoenzymes were identified by the combination of the selective CYP inhibitor study, correlation analysis and a panel of recombinant rat CYP expereiment, respectively. RESULTS: The results from the experiments of selective CYP inhibitors, correlation analysis and recombinant rat CYP isoenzymes indicated that CYP2A1, CYP3A and CYP2C11 contributed to the formation of M1 and M5 in the rat liver microsomes. CYP3A, CYP2A1, CYP1A2, CYP2C11 and CYP2E1 metabolized Dip to M2. CYP3A, CYP2A1, CYP2E1 and CYP2C11 contributed to M4 formation. And the recombinant rat CYP researches further indicated that CYP3A2 exhibited more activity than CYP3A1. CONCLUSION: CYP3A and CYP2A1 are the major CYP isoenzymes responsible for catalyzing Dip to the four metabolites formation in the rat liver microsomes.
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OBJECTIVE: To evaluate the effects of dipfluzine hydrochloride (Dip) on CYP450s activities in vitro and in vivo in rats. METHODS: Markers were incubated in the normal rat liver microsomes with Dip (0-200 μmol·L-1) and the concentration of metabolites of the markers were determined by LC-MS/MS, and then the ratios were calculated to evaluate the effects of Dip on the CYP450s activities. Dip was administered by orally to the male SD rats at doses of 30, 60 and 90 mg·kg-1 body weight and phenobarbital was administered at doses of 120 mg·kg-1 body weight for 14 d. At the fifteenth day, the rats were orally administered the Cocktail probe markers and blood samples were collected via medial angle of eye at different time. The concentrations of markers were determined by LC-MS/MS and the drug-time curve was plotted, by which the pharmacokinetic parameters were calculated. And then the rat liver microsomes were prepared and the probe markers were added into the incubation samples. The concentrations of the probe markers and its metabolites were determined and the metabolism ratios were calculated. The effects of Dip on CYP450s activities were evaluated by comparing the following outcomes between the experimental groups and the control group: the relative liver weight, the concentrations of protein, the contents of CYP450 enzymes, the drug concentration-time curves, the pharmacokinetic parameters and the metabolism ratios of the probes. RESULTS: In the normal rat liver microsomes, Dip had the inhibitive effects on CYP2D1, CYP2C6 and CYP2C11, and the IC50 were 8.85, 20.93 and 69.45 μg·mL-1, respectively. Dip had no effect on the relative weight of livers, the protein concentrations and the CYP450 content for the rats after they were fed on Dip for 14 d, but these indexes were raised remarkably when phenobarbital was administered by orally to rats. The results displayed that the low-dose Dip inhibited the activity of CYP2D1 or induced the activity of CYP2C11 in rats. Moderate-dose Dip showed the abilities to inhibit CYP2D1, but induce CYP2C11 and CYP3A. High-dose Dip had the certain inhibitive effects to CYP2C6 and CYP2D1, and had the inductive effects on CYP2C11 and CYP3A. CYP1A2, CYP2C11, CYP2C12, CYP2D1 and CYP3A were all induced after administration of phenobarbital. CONCLU SION: The results of liver microsomes incubation and blood plasma from the normal and Dip-induced rats all show that Dip inhibit the activities of CYP2C6 and CYP2D1, however, Dip inhibit CYP2C11 in vitro and induce it in vivo.
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OBJECTIVE: To establish a robust, fast and convenient method for in vitro assay of rat liver CYP1A2 and CYP2D1, and explore their kinetic features. METHODS: Two selective substrates including phenacetin and dextromethorphan, which are probes of CYP1A2 and CYP2D1, were chosen for liver microsomes incubation, respectively; the corresponding ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS) methods were developed for kinetic studies. RESULTS: The fast and convenient UPLC-MS methods with high resolution and short running time (4-5 min) were established and validated for two assays of CYP1A2 and CYP2D1 activities; both methods showed good accuracy and precision, and the values of LOQ for CYP1A2 and CYP2D1 assays could reach 0.267 and 0.007 μmol·L-1, respectively. The kinetic studies showed that the Michaelis constant (Km) for CYP1A2 and CYP2D1 were (28.4±2.7) and (13.9±1.3) μmol·L-1, respectively. Their activities were determined to be (1.47±0.12) and (3.98±0.09) nmol·mg-1, respectively, when the substrate concentration was 10 μmol·L-1. CONCLUSION: UPLC Tandem MS technique is proved to be a rapid, convenient and efficient approach with high sensitivity and selectivity for the assays of CYP1A2 and CYP2D1 in drug metabolism.
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OBJECTIVE: To study the induction effect of bencycloquidium bromide (BCQB) on rat liver cytochrome P450 enzymes. METHODS: Rats were divided into solvent control group, positive control (phenobarbital) group and BCQB group. The rats in BCQB groups were intranasally administered with 1, 3, and 9 mg · kg-1 BCQB, respectively. After multiple-dose administration, the rats were sacrificed, and the livers were weighed and prepared to microsomes. Then the liver coefficients were calculated, and the total content of CYP450 enzymes in liver microsomes was determined by spectrophotometer. HPLC-MS/MS method was adopted and validated to simultaneously determine the productive velocity of 6β-hydroxyl testosterone and paracetamol after incubation of rat liver microsomes. The activities of CYP1A1/2 and CYP3A1/2 were measured according to the productive velocity. RESULTS: The liver coefficients, total content of CYP450 enzymes, and activity of CYP1A1/2 were significantly different (P0.05) between solvent control group and BCQB groups. The activity of CYP3A1/2 in BCQB groups was not higher than solvent control group. CONCLUSION: Phenobarbital has induction effect on rat liver CYP450 enzymes. The test system can be used to evaluate the induction effect of BCQB on rat liver CYP450 enzymes. BCQB has no induction effect on liver CYP450 enzymes in rats.