Dissertation > Medicine, health > Chinese Medicine > Of Pharmacy > Pharmacology

Effects of Five 6, 7-Furocoumarins on the Hepatic Microsome Cytochrome P450 1A2, 2E1 Activity in Rats

Author LiChao
Tutor WangDeCai
School Taishan Medical College
Course Pharmacology
Keywords 6, 7-Furocoumarin compounds Cytochrome P450 1A2 Cytochrome P450 2E1 Caffeine Chlorzoxazone Drug metabolism
CLC R285
Type Master's thesis
Year 2009
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Objective:To investigate the effect of five 6,7-furocoumarin compounds(psoralen、bergapten, isopimpinellin, imperatorin, isoimperatorin)on hepatic microsome CYP1A2 and CYP2E1 activity in rats , to compare the potency of the five compounds, and to provide theoretical basis for rational use of clinical drug and new drug development.Method:①Hepatic microsomes were prepared by the CaCl2 precipitation method in healthy adult male Wistar rats, and then measured the protein concentration, contents of CYP and Cytb5 in hepatic microsome suspension.②Caffeine and chlorzoxazone were used as probe drugs of CYP1A2 and CYP2E1, antipyrine was used as internal standard, simultaneous detected concentration changes of two probe drugs in reactive system in vitro by HPLC and carried on the method investigation.③The microsomes suspension, Tris buffer and accessory factor solution were composed of a reactive system in vitro. The NADPH which probe drugs metabolism needed was produced by NADP+, G-6-PD, G-6-P, MgCl2 in accessory factor solution. Optimization of reaction conditions carried on in 37℃incubation time, the concentrations of hepatic microsome protein and probe drugs, and determined the best conditions.④The terminal concentrations of five 6, 7-furocoumarin compounds and positive control chloramphenicol were 1.0, 2.0, 4.0, 8.0, 16.0μg·mL-1 in vitro, also seted up the blank tube/group, after added probe drugs in tube, to incubate in water for 40 minutes at 37℃, and stoped reaction by ice-cold chloroform. The concentrations of probe drugs were detected with HPLC, and the probe drug conversion rate was calculated. Repeated operation 6 times. To evaluate the effects of five 6, 7-furocoumarins on CYP1A2 and CYP2E1 activity through the differention of the conversion rate.Results:①The concentration of protein was (18.265±2.72)mg·g-1 liver wet, contents of total CYP and Cytb5 were (0.439±0.156)nmol·mg-1, (0.234±0.112) nmol·mg-1,respectively.②Chromatographic conditions: mobile phase: methanol: water 30:70; flow rate: l.0ml·min-1; column temperature: 25℃; detective wavelength: 280nm. Caffeine, chlorzoxazone and antipyrine were separated completely, and retention times were 3.81, 21.50 and 6.25minuts, respectively. Satisfactory linear relationship was achieved when the mass concentration of caffeine was within a range of 0.520.0μg·mL-1 (r=0.9998), within-day RSD≤5.26%, between-day RSD≤4.52%; the recovery rate of lower, middle and higher concentration of caffeine was 105.0%, 96.4%, 97.1%, respectively. There was a good linear relationship between the ratio of peak area and the content of chlorzoxazone in a range of 0.5 20.0μg·mL-1 (r= 0.9995), within-day RSD≤5.91%, between-day RSD≤1.90%; the recovery rate of lower, middle and higher concentration of chlorzoxazone was 96.0%,101.6%,90.16%,respectively.③The reaction conditions in vitro: incubation temperature was 37℃, incubation time was 40min, the concentration of microsome protein was 1.0mg·ml-1, the terminal concentrations of caffeine and chlorzoxazone were 8.0μg·ml-1.④The conversion rate of caffeine and chlorzoxazone were (63.8±5.1)% and (43.5±7.6)% respectively in the blank control group. Chloramphenicol could inhibit significantly two probe drugs conversion rate(all P<0.05)at its five concentrations and were fitted well in a dose-dependent manner. Two probe drugs conversion rate had the varying degree reduction in the five reagent drug groups, compared with blank group, there was statistical significance except a few lower concentrations, and there were showed dose-effect relation in all. Under the same mass concentration, caffeine conversion rate at 2.0, 4.0, 8.0μg·mL-1of isoimperatorin was lower than chloramphenicol(P<0.05); chlorzoxazone conversion rate had significantly decreased at 2.0,4.0,8.0μg·mL-1 of imperatorin or bergapten was lower than chloramphenicol(P<0.05); the conversion rate of two probe drugs was higher at all concentrations of isopimpinellin, compared with chloramphenicol(P<0.05).Conclusions:①Hepatic microsome suspension which was prepared by the CaCl2 precipitation method could meet the need of drug metabolic research in vitro.②Antipyrine was used as internal standard. Seted up a reversed-phase high performance liquid chromatography (RP-HPLC) method which simultane- ously detected caffeine and chlorzoxazone. This detective method was simple operation , and its sensitivity, reliability and stability were all satisfactory.③The inhibition of five 6, 7- furocoumarin compounds on CYP1A2 and CYP2E1 was dose-dependent, and the inhibition on CYP1A2: isoimperatorin > imperatorin > psoralen≥bergamotine > isopimpinellin, the inhibition on CYP2E1: imperatorin≥bergapten > isoimperatorin≥psoralen > isopimpinellin. The inhibitive activities of isoimperatorin on CYP1A2 and bergapten on CYP2E1 were probably higher than chloramphenicol, but the effects of isopimpinellin was lower than chloramphenicol.

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