金果榄中古伦宾在家兔体内的药动学研究

Pharmacokinetic study of columbin from Radix Tinosporae in rabbits in vivo

目的研究金果榄中古伦宾在家兔体内的药动学。方法从金果榄的干燥块根中制备提取古伦宾,建立超高效液相色谱(UPLC)检测古伦宾方法。色谱条件,色谱柱:Acquity UPLCRBEN C18(2.1×100 mm,1.7μm);流动相:0.3%磷酸溶液-乙腈(65:35);流速:1 ml/min;检测波长:225 nm;柱温:35℃。考察该方法的专属性、线性、定量限、提取回收率、精密度和稳定性;测定不同浓度古伦宾对照品标准溶液的峰面积,制作古伦宾浓度标准曲线;对家兔进行古伦宾灌胃注药(50 mg/kg),于给药后0、15、30、60、120、180、240、360、480、1400 min后进行耳缘静脉采血,测量古伦宾浓度,绘制血药浓度-时间曲线,运用Win Nonlin 3.3软件计算相应古伦宾在家兔体内的药动学参数。结果色谱图峰形显示,古伦宾保留时间在16 min,峰形正态分布,与其他峰分离良好,无内源性物质干扰。古伦宾浓度标准曲线方程为Y=502967X+0.1501,相关系数r=0.995,线性范围为0.00122~2.50000 nmol/L,最低定量限为1.22×10-3nmol/L。质量浓度为9.77、156.25、1250.00 nmol/L的古伦宾血浆溶液提取回收率均在80.0%以上,平均回收率为84.2%,相对标准偏差(RSD)=3.1%。156.25 nmol/L的古伦宾血浆溶液的日间RSD=3.5%。第1天日内RSD=1.7%,第2天日内RSD=4.6%。室温保存24 h,反复冻融3次,以及-20℃冷冻保存14 d时的回收率均为90.0%~110.0%。药动学参数:达峰时间(Tmax)=68.9 min、峰浓度(Cmax)=0.0671 nmol/L、末端消除半衰期(T1/2)=438.67 min、药时曲线下面积(AUC)=81.49、末端消除速率(Ke)=0.0017、清除率(CL)=20132.72、生物利用度(F)=14.08%。结论 UPLC法测定古伦宾浓度,其专属性、提取回收率、稳定性均符合要求,适用于对金果榄中古伦宾在家兔体内的药动学进行系统研究。

Objective To study the pharmacokinetics of columbin from Radix Tinosporae in rabbits in vivo. Methods The columbin was extracted from the dried root of Radix Tinosporae, and the UPLC method for the determination of columbin was established. Chromatographic conditions, column： Acquity UPLCRBEN C18（2.1×100 mm, 1.7 μm）; mobile phase： 0.3% phosphoric acid solution-acetonitrile（65：35）; flow rate： 1 ml/min; detection wavelength： 225 nm; temperature： 35°C. The specificity, linearity, quantitative limits, extraction recovery, precision and stability of the method were investigated. The peak area of the standard solution of different concentrations of culumbin was determined, and used to make the standard curve of the concentration of culumbin. The culumbin（50 mg/kg） was gavaged to rabbits,and limbic vein blood collection was conducted at 0, 15, 30, 60, 120, 180, 240, 360, 480 and 1400 min after administration. The blood concentration-time curve was drawn, and the corresponding pharmacokinetic parameters of culumbin in rabbits were calculated by using Win Nonlin 3.3 software. Results Peak shape of the chromatogram showed that the retention time of culumbin was 16 min, with normal peak shape distribution. It was well separated from other peaks,and there was no endogenous material interference. The standard curve equation of culumbin concentration was Y =502967 X＋0.1501, the correlation coefficient r=0.995, the linear range was 0.00122-2.50000 nmol/L, and the minimum quantitative limit was 1.22×10-3 nmol/L. The recovery rate of the plasma solution of culumbin at the mass concentration of 9.77, 156.25 and 1250.00 nmol/L was over 80.0%, the average recovery rate was 84.2%, and the relative standard deviation（RSD）=3.1%. The RSD between two days of the 156.25 nmol/L culumbin plasma solution was 3.5%, the first day RSD was 1.7%, the second day RSD was 4.6%. Preserved 24 h at room temperature, repeated freezing,thawing 3 times, and-20℃ cryopreservation 14 d average recovery was 90.0%-110.0%. Pharmacokinetic parameters：Tmax=68.9 min, peak concentration（Cmax） =0.0671 nmol/L, terminal elimination half-life（T1/2）=438.67 min, medication in the area under the curve（AUC）=81.49, end eliminate rate（Ke）=0.0017, clearance（CL）=20132.72, bioavailability（F）=14.08%. Conclusion The UPLC method was used to determine the concentration of columbin, and its specificity, extraction recovery and stability all meet the requirements, which is suitable for the systematic study of the pharmacokinetics of columbin in rabbits.