| OBJECTIVE To study the inhibition of effective composition of five traditional Chinese medicine (scutellarin, picrinine, cucurbitacin IIa, swertiamarin, gastrodin) and the preparation of scutellarin on CYP1A2 activity in rats in vitro and in vivo.METHODS (1) BCA, Bradford and Lowry methods were used to examine their sensitivity, accuracy, precision, linear range and impact factors, as well as the concentration of rat liver microsomes protein. (2) The constitution of the incubation system was optimized and enzymatic kinetic parameters were analyzed. (3) To study the affection of phenacetin with or without effective composition of five traditional Chinese medicine in Yunnan, and calculated IC50 values. (4) Combined experiments to study the effect of scutellarin preparations on CYP1A2 activity in vitro and in vivo. In vitro, study the affection of phenacetin with or without scutellarin preparation from different manufacturers, and IC50 was calculated. In vivo, metabolic of phenacetin was determined in 7-days-pretreated scutellarin preparations group. Relevant pharmacokinetic parameters were calculated by using DAS 2.11 software.RESULTS (1) The three methods have good correlation and linearity. BCA method had the largest linear range. The precision and accuracy of Lowry method and BCA method were superior to the Bradford method, and resistant ions interference is significantly better than Bradford method. (2) The optimum incubation time was 20 minutes, incubation temperature was 37℃, the concentration of protein in rat liver microsomes was 0.8mg/mL, the concentration of best probe substrate was 75μmol/L, the best use of time was 3 months. Km was 73.05±1.54μmol/L and Vmax was 243±83pmol/min/mg protein. (3) IC50 of scutellarin and picrinine was 108.20±0.657 and 118.559±8.659μmol/L respectively; IC50 of cucurbitacin IIa, swertiamarin and gastrodin was 509.401±10.12,173319.2±14061.24 and 18275.7±4716.66μmol/L respectively. (4) In vitro experiment, IC50 of Longjin, Peisiting Shenwei, Longtai, Hongfeng scutellarin preparation was 240.94±6.65,248.58±5.77,141.40±3.18, 4597.1±1205.3 and 458.01±5.70μg/mL respectively. After given 7 days of scutellarin preparation, the AUC0-∞, half-life and clearance rate was 1665.88±501.49mgxmin/L,37.86min and 0.01L×kg/min respectively, which were statistically significant (p<0.05).CONCLUSION (1) The Lowry method is easy to operate, which is more suitable for laboratory research. The advantages include economical raw material, higher accuracy and stability. (2) By examining enzymatic kinetics of phenacetin, the specific substrate of rat CYP1A2, to offer reference and support in vitro study. (3) Scutellarin and picrinine have a weak inhibition on CYP1A2 in rats. It is possible to lead drug interactions when associated with the drug metabolism by CYP1A2. It should be carefully used and strictly monitored. (4) Scutellarin production from different manufacturers showed different inhibition activity on CYP1A2 in vitro study, which may be caused by difference scutellarin content and different excipients in preparation; after 7-days-pretreated scutellarin preparation, it showed a weak inhibition on CYP1A2 activity, which slow down the metabolism/eliminate of phenacetin. In order to prevent drug interactions and risk of the combined using drugs, the dosage of drugs metabolized by CYP1A2 should be strictly controlled and monitored when given scutellarin preparation treatment. |
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