四逆汤有效部位的药代动力学——药效动力学研究

【作者】 晏亦林；

【导师】 李锐； 周莉玲；

【作者基本信息】 广州中医药大学 ， 中药学， 2001， 硕士

【摘要】 四逆汤始载于《伤寒论》，由附子、干姜、炙甘草组成，方中附子“味辛大热，纯阳有毒，其性走而不守”，干姜“大热无毒，守而不走”，二阳相合，一走一守，佐以甘草，具有回阳救逆之功效，临床上常用于阳虚欲脱、脉微欲绝等症。 目的：对四逆汤有效部位进行药代动力学（Pharmacokinetics，PK）-药效动力学（Pharmacodynamics，PD）研究，阐明四逆汤回阳救逆的物质基础，揭示其“走而不守，守而不走”的配伍规律。 方法：（1）四逆汤的制备：分别提取附子、干姜、炙甘草，合并。（2）四逆汤的精制：应用D₁₀₁大孔吸附树脂精制。（3）四逆汤精制物的药学评价：用薄层层析法（TLC）考察附子中的生物碱类成分，甘草中的甘草酸、甘草黄酮类成分及干姜中的一些特征成分；用高效液相色谱法（HPLC）考察乌头类生物碱（包括双酯型生物碱及其水解产物）的指纹图谱特征并测定其含量。（4）四逆汤精制物的药效学评价：选择离体蛙心的心肌收缩力、心输出量、心率等强心指标及促进血管扩张，改善休克状态的一氧化氮（NO）指标。（5）血药浓度HPLC测定方法的建立。（6）PK-PD研究：取狗肌内注射四逆汤精制物，分别于给药前、给药后不同时段股动脉取血，取同一时相的血清测定乌头类生物碱含量及NO含量；所得实验数据用MCPKP自动化药物动力学程序软件处理，计算药动学参数；进行相关性研究。 结果：（1）四逆汤精制的条件：四逆汤上柱药液的浓度为0.5g/ml，乙醇洗脱浓度为70％，流速为1ml/min。在此条件下，乌头类生物碱的解吸率为83.57％，干膏量为总量的5.23％。（2）药学研究结果表明：与四逆汤比较，四逆汤精制物TLC图谱中斑点个数、大小及颜色均基本相同，保留了附子中的生物碱类成分，甘草中的甘草酸及甘草黄酮类成分，干姜中的一些特征成分。四逆汤精制物HPLC图谱中乌头类生物碱及其水解产物峰的峰数、峰位、峰形均与四逆汤基本一致。乌头类生物碱的含量为0.0243mg/ml。（3）药效学实验证明：四逆汤精制物广州中医药大学硕士学位论文能增加离体蛙心心肌收缩力及心输出量，减慢心率；显著升高间羟胺处理小鼠血清中NO的含量，与四逆汤相比无显著性差异。（4）血药浓度HPLC测定方法的研究表明：血清中乌头碱对照品的线性方程为：Y＝42667．11982X－2817．133，相关系数 r＝0．9995，线性范围为：1—20u g／ml。高、中、低三个浓度的乌头碱对照品液天内回收率分别为93．60％、94．56％、94．76％，RSD分别为3．20％、2．62％、2．44％；天间回收率分别为 93．30％、93．99％、93．09％，RSD分别为 2．60％、1．54％、2．11％。 门)PK干D研究结果表明：乌头类生物碱血药浓度及NO净增率在狗体内的动态变化均符合开放一房室模型。血药浓度法所测参数为：K。二4．94052bYS－l，K二0．32852 hTS－l，TI／ZK。二0．144llhYSITI／ZK二2·11256hi’S，Tp二0．59622hi’S，CB．：二2．21856 119加lAUC二8．2332Sg．hgs／l，V＝0．029961／kg CI二0．009861／kg．hgs；药理效应 法 所 钡 参 数 为：K。＝4．16196hSS－‘，K二0．43185hrs－‘T；／、。。二0·16942hrs‘，T；；。。二1．60573hrs，Tp＝0．61320hrs。在 0一6hr之间乌头类生物碱血药浓度与NO净增率存在良好的相关性。相关方程为：Y＝0．4478X－0．1123，r＝0．9939o 结论：乌头类生物碱的体内动态变化在一定时间范围内可以反映四逆汤升高血清NO含量的药效程度，血药浓度法与药理效应法在一定程度上存在良好的相关性，乌头类生物碱是四逆汤回阳救逆的物质基础；药动学参数表明四逆汤有效部位起效快，维持时间长，与四逆汤“走而不守，守而不走”的配伍规律相吻合。更多还原

【Abstract】 AbstractSini Decoction(SND), from Treatise on Exogenous Febrile Diseases, iscomposed of Asdix Aconiti Praeparata, Anizoma Zingiberis and RadixGlycyrrhizae Praeparata. Aconiti, heavy acrid fiavour and great hot nature,has the effect of warming and tonifying kidneym. Its effect is pompt butnot lasting. Zingiberis can warm and tonify spleenW and its effect islasting but not prompt. The combination of these two drugs, assisted byglycyrrhizae gives a prompt and lasting effect. Sini Decoction canrecuperate the depleted yang and rescue the patient from danger, main1y forcases of exhaustion of yang-energy, manifested as deadly cold of limbs,sunken and feeble pulse.Objective: to study the effective fraction of SN’D bypharmacokinetics(PK)-pharmacody method, to study thepharmacological substance of SND, to reveal the comPatibility law of SND.Methods: (l) Preparation of SNDf Radix Aconiti Praeparata,Rhizoma Zingiberis and Radix Glycyrrhizae Praeparata are extractedrespectively, merged into SND. (2) Purification of SNDf SND is purified bymacroporous resin D,,,’ (3) Pharmaceutical evaluation of the purification fThe alkaloids in Radix Aconiti Praeparata, glycyrrhizic acid, liquiritigenin,isoliquiritigenin in Radix Glycyrrhizae Praeparata, and some markercomponents in Rhizoma Zingiberis are identified by TLC(Thin LayerChromatography). The fingerprint chromatograms and content of aconitine-type alkaloids(including doub1e ester-type alkaloids and their hydrolysates )are inspected and determined by HPLC (High Pefformance LiquidChromatography). (4) Pharmacological evaluation of the purification f Theindexes include myocardiac contraction, cardiac output, heart rate of frog’s3separated heart and the content of nitric oxide (NO), which cal1 dilate bloodvessel and remedy shock. (5) The method of determining the pIasl11aconcentration by HPLC is developed. (6) PK-PD study f Three dogs areintramuscular injected with the purification. The bIood is got from thighartery befOre the injection and at different moments after injectio11. Thecontents of aconitine-type alkaloids and NO of the same moment’s serumare determined. After the data are processed by MCPKI, PharmacokineticPC Program, the parameters are caculated and the correlation is studiedbetween the plasma concentration and the NO net growth rate duri11g 0~6hr.Results: (l) The optimum factors of purifing SND’ The concentrationof SND extraction is 0.5g/ml, the eluating ethanol concentration is 70%, andthe flow rate is lml/min.The absorption recovery of aconitine-type alkaloidsis 83 .97%, while the weight of the dried purification is 5.23% of the total. (2)The pharmaceutical study shows the thi1l layer chromatograms of the SNDpurification and those of SND are almost the same. The purification keepsaconitine-type alkaloids in Radix Aconiti Praeparata, glycyrrhizic acid,Iiquiriti8enin, isoliquiritigenin in Radix Glycyrrhizae Praeparata and markercomponents in Rhizoma Zingiberis. High performance liquid chromatogran1of the purification is basically the same as that of SND, while the co11tent ofaconitine-type alkaloids is 0.0243mg/ml determined by HPLC. (3) TI1epharmacoIogical experiments proved that the SND purification can promotemyocardiac contraction and cardiac output, lower the heart rate of frog’sseparated heart, raise siSniflcantly the content of NO in serum of micetreated by Metaraminol. (4) The regression equation of acol1itine refCrel1cestandard in serum determined by HPLC is Y=42667. l l982X-28l7. l33, r=0 9995, the linear range is l~20 P g/ml. The precision and accuracy of t11emethod can meet the needs of the experiment. (5) The data of PK and PDcan be botl1 described by one compartme11t open model witl1 first order4absorption. The parameters of two methods are similar. The regressionequation of the plasma concentration and the NO net growth rate during0--6hr is Y=0.4478X-0.ll23, r=0.9939, showing the corre更多还原