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小檗碱对大鼠体内葛根素药动学的影响
Effect of Berberine on Pharmacokinetics of Puerarin in Rats
【作者】 吴恒；
【导师】 陈礼明；
【作者基本信息】 安徽医科大学 ， 中药学， 2010， 硕士
【摘要】 目的:建立大鼠血浆中葛根素（Puerarin）浓度的高效液相色谱（HPLC）测定法,观察葛根素在大鼠体内的药动学过程以及小檗碱对大鼠体内葛根素药动学的影响,并计算相应的药动学参数,探讨两药合用后葛根素在大鼠体内的药动学变化情况。方法:血浆样品采用6%高氯酸沉淀蛋白,15000 r·min-1高速离心后进行检测。色谱柱为Hypersil ODS（4.6 mm×150 mm,5μm）,流动相为甲醇-0.0043mol·L-1磷酸二氢钾缓冲液（pH2.7）（22:78）,流速为0.8 mL·min-1,柱温为30℃,检测波长250 nm,进样量为20μL。大鼠灌胃给予葛根素混悬液(100 mg·kg-1 )及葛根素和小檗碱混合液(100 mg·kg-1 + 50 mg·kg-1、100 mg·kg-1 + 100 mg·kg-1、100 mg·kg-1 + 200 mg·kg-1),用HPLC法测定大鼠给药后不同时间血浆葛根素的浓度,所得药物浓度-时间数据用DAS 2.1.1程序处理,进行自动房室模型判别及药动学参数计算,并对单独用药组及联合用药组进行比较。结果:1.葛根素的标准血药浓度的线性范围为0.10～10.00 mg·L-1,回归方程为A = 2.7019×104 C + 2495.8 ,r = 0.999 5,葛根素的最低检测限（LLOD）为0.05 mg·L-1 （S/N>3）;高、中、低三种浓度的日内、日间精密度RSD分别小于2.11%和2.79% ;方法回收率为96.64%～112.55%。2.合用小檗碱前后,葛根素混悬液组(100 mg·kg-1)及葛根素和小檗碱混合液组(100 mg·kg-1 + 50 mg·kg-1、100 mg·kg-1 + 100 mg·kg-1、100 mg·kg-1 + 200 mg·kg-1)四组中葛根素的主要药动学参数Cmax分别为（0.545±0.051）mg·L-1、（0.587±0.030）mg·L-1、（0.667±0.017）mg·L-1、（0.733±0.0298）mg·L-1;AUC0～t分别为（2.019±0.388）mg·L-1·h、（1.962±0.254）mg·L-1·h、（2.008±0.619）mg·L-1·h、（2.969±0.295）mg·L-1·h;CL分别为（32.937±12.695）L·h-1·kg-1、（32.656±3.599） L·h-1·kg-1、（34.556±9.167）L·h-1·kg-1、（22.748±4.654）L·h-1·kg-1。结果显示葛根素与小檗碱合用后,葛根素和小檗碱混合用药组(100 mg·kg-1 + 50 mg·kg-1)药动学参数与葛根素组(100 mg·kg-1)无显著性差异;葛根素和小檗碱混合用药组(100 mg·kg-1 + 100 mg·kg-1、100 mg·kg-1 + 200 mg·kg-1) Cmax增大,有显著性差异（P < 0.01）;葛根素和小檗碱混合用药组(100 mg·kg-1 + 200 mg·kg-1) Cmax,AUC0～t均增大,有显著性差异（P < 0.01）。结论:本实验建立的大鼠血浆中葛根素HPLC测定法,操作简单,费用低,方法可靠,灵敏度高,血浆中成分不干扰,能较好适应目前大多数实验室条件,适用于葛根素的血药浓度检测及药动学研究。大鼠灌胃葛根素后的药-时过程符合二室模型,与国内外文献相近,葛根素在动物体内的药动学过程的研究,有助开展中药复方中主要有效成分间的配伍研究,探讨中药复方内在配伍规律。
【Abstract】 OBJECTIE:To establish a sensitive and specific method for determination of puerarin in rat plasma by HPLC and study its pharmacokinetics.METHODS:Puerarin was extracted from plasma with perchloric acid and was centrifugated by high speed. HPLC analysis was carried out through Hypersil ODS column（ 4.6 mm×150 mm, 5μm ）.The mobile phase consisted of methanol-0.0043mol·L-1 potassium dihydrogen phosphate （ 22:78 ） at a flow rate of 0.8 mL·min-1. The column temperature was 30℃and the wave length was 250 nm. Plasma puerarin concentration was determined by HPLC after intragastrically given of puerarin ( 100 mg·kg-1 ) or puerarin combined with berberine ( 50mg·kg-1, 100mg·kg-1, 200mg·kg-1 ) in rats. The data obtained were processed by DAS 2.1.1 software to calculate the varities of pharmacokinetic parameters after ig single durg or mixed drugs. RESULTS: 1. The standard curve was linear in the range of 0.10 mg·L-1～10.00 mg·L-1（ A = 2.7019×104 C + 2495.8, r = 0.999 5 ）. The lowest limit of detection was 0.05 mg·L-1. The relative standard derivation of inter-day and intra-day was smaller than 2.11% and 2.79%. The relative recoveries of Puerarine were 96.64%～112.55%.2. The main pharmacokinetic parameters of puerarin before and after coadministmtion of berberine were as follow: Cmax were 0.545±0.051 mg·L-1, 0.587±0.030 mg·L-1, 0.667±0.017 mg·L-1, 0.733±0.0298 mg·L-1; AUC0～t were 2.019±0.388 mg·L-1·h, 1.962±0.254 mg·L-1·h, 2.008±0.619 mg·L-1·h, 2.969±0.295 mg·L-1·h; CL were 32.937±12.695 L·h-1·kg-1, 32.656±3.599 L·h-1·kg-1, 34.556±9.167 L·h-1·kg-1, 22.748±4.654 L·h-1·kg-1. The results showed that, there was no significant difference in other pharmacokinetic parameters of puerarin between Group 1 and Group 2. While there were no significant differences for pharmacokinetic parameters of puerarin except increasing of Cmax after oral doses ( 100mg·kg-1 berberine, 200mg·kg-1 berberine ) （ P < 0.01 ）. AUC0～t of puerarin increased in Group 4 compared with Group 1.CONCLUSIONS:The method was specific, sensitive and simple, and suitable for determination of Puerarin in rat plasma and pharmacokinetics researches. The results were accurate, a two-compartment pharmacokinetic model was adapted to Puerarin plasma concentration-time data analysis, the main pharmacokinetic parameters were similar to those reported domestic and abroad. So it could assist to approach internal compatibility in traditional Chinese medicine complex prescription.