【作者】 杜玮；

【导师】 武新安；

【作者基本信息】 兰州大学 ， 药物分析学， 2008， 硕士

【摘要】 目的:（1）分别建立简单、灵敏的反相高效液相色谱方法（RP-HPLC）,对丹参酮Ⅰ以及丹参醇提物中多组分在大鼠的血药浓度进行测定,并将其应用于药动学研究中。（2）考察复方丹参片对大鼠的肝药酶CYP450的影响。（3）建立一种高效液相色谱-二极管阵列检测器-蒸发光散射检测器联用技术,用于复方丹参片中多元组分的定量测定。并结合化学计量学中的模式识别技术对复方丹参片进行质量评价方法:（1）采用Cosmosil C₁₈column（4.6mm×150mm,5μm）,流动相采用乙腈-0.02 mol·L^-1乙酸铵缓冲液-三乙胺（66:34,v/v）,流速为1.0 mL·min^-1,检测波长为263nm。通过股动脉插管的给大鼠瞬间注射丹参酮Ⅰ的生理盐水后,采集血样,用含有内标10μg·mL^-1苯甲酸雌二醇（内标）的乙腈沉淀血浆蛋白。采用反相高效液相色谱方法测定血浆中丹参酮Ⅰ的浓度,并用DAS1.0软件估算相应的药动学参数。采用VP-ODS C₁₈柱（4.6 mm×250 mm;5μm）,以甲醇-0.05 mol·L^-1醋酸铵缓冲溶液（醋酸调pH至2.5）（70:30,v/v）为流动相,流速1.0 mL·min^-1,检测波长263 nm,柱温40℃,以丙酸睾丸素为内标,给大鼠灌胃丹参的醇提取物后,通过股动脉插管的方式采集不同时间的血样,测定血浆四种丹参酮的浓度,并估算药动学参数。（2）以非那西丁为探针药物,四组大鼠分别灌胃给予生理盐水（空白对照组）、西咪替丁（酶抑制剂组）、苯巴比妥钠（酶诱导剂组）和复方丹参片（受试药物组）后,均连续给药7天,于第8日早晨腹腔注射探针药物非那西丁,于不同时间点采集血样,用HPLC法测定血浆中探针药物浓度,用DAS软件估算各组探针药物药动学参数。（3）应用HPLC-DAD-ELSD联用技术,根据各类成分紫外吸收光谱的差异,分别在287,246,263,292和268nm波长下检测丹酚酸B、二氢丹参酮、隐丹参酮、丹参酮Ⅰ、次甲基丹参酮和丹参酮ⅡA,同时采用ELSD对5中皂苷（三七皂苷R1、人参皂苷Rg1、Re、Rb1和Rd）进行测定。结果:（1）丹参酮Ⅰ在高、低浓度范围:(0.1-1.0μg·mL^-1、1.0-10.0μg·mL^-1)的线性方程分别为Y=0.8584X-0.0367和Y=0.9424X-0.4675,其相关系数r²均为0.9998,日内日间准确度均高于91.33%,精密度RSD均小于11.0%,回收率大于88.3%。在信噪比为10时,丹参酮Ⅰ的最低定量限为10 ng ml^-1。大鼠静脉注射丹参酮Ⅰ的药代动力学特征符合二室开放模型,呈一级动力学消除,属于吸收快,消除较慢的过程。隐丹参酮、丹参酮Ⅰ、次甲基丹参酮和丹参酮ⅡA的线性范围分别为:0.40-25.5μg·mL^-1;0.51-32.5μg·mL^-1;0.40-27.5μg·mL^-1;0.42-26.5μg·mL^-1,其回收率均大于88.7%。药代动力学参数表明:大鼠灌胃丹参醇提取物后,隐丹参酮和丹参酮Ⅰ的药代动力学特征符合一室模型,在体内吸收后迅速分布;次甲基丹参酮和丹参酮ⅡA的药代动力学特征符合二室模型,在体内吸收后分布缓慢。四种丹参酮中,隐丹参酮消除最慢,丹参酮Ⅰ次之,而丹参酮ⅡA的消除最快。（2）生理盐水组、西咪替丁组、苯巴比妥钠组和复方丹参片组探针药物的t_1/2分别为93.51±9.21,161.67±10.95,85.36±8.98和77.93±6.54分钟。复方丹参片组探针药物的t_1/2为77.93 min,与生理盐水组进行统计分析有统计学意义（t＜0.01）。因而可以初步推断复方丹参片对大鼠肝药酶P450具有一定的诱导作用。（3）测定了4个不同厂家的15批复方丹参片成品中11种活性成分的含量,并采用主成分分析、聚类分析法进行质量评价,得出生产工艺和药材产地为影响药品质量的主要因素。结论:（1）建立的高效液相色谱法简便、可靠、准确,可用于生物样品中丹参中多组分的血药浓度分析和药代动力学研究。丹参酮Ⅰ和次甲基丹参酮的药动学过程尚属首次报道。（2）由于复方丹参片在临床上使用广泛,且用药周期长,因此提示:在和经肝药酶代谢的西药联合使用过程中,应注意中西药的合理配伍。（3）本实验建立的HPLC-DAD-ELSD联用技术和化学计量学相结合的方法能对中药复方制剂的质量进行科学的评价,该方法为中药复方复杂体系的多组分定量测定和质量控制提供了一种可靠、简便易行的方法模式。创新性:（1）首次建立了同时测定大鼠血浆中四种丹参酮浓度的RP-HPLC方法,其中丹参酮Ⅰ和次甲基丹参酮在大鼠体内的药代动力学研究尚属首次。（2）首次研究了复方丹参片对大鼠肝药酶CYP450的影响。（3）首次采用HPLC-ELSD联用技术,测定了复方丹参片中的11个活性成分,并结合化学计量学的模式识别方法,全面系统的评价了复方丹参片的质量。更多还原

【Abstract】 OBJECTIVE:（1）To develop a new reversed phase high performance liquid chromatographic method（RP-HPLC）for determining the concentration of TanshinoneⅠin rat plasma following intravenous injection of TanshinoneⅠsolution, and the pharmacokinetic parameters of TanshinoneⅠwas caculated for the first time by Drug and Statistics 1.0 program.To develop A for simultaneous determinination of four tanshinones in rat plasma was developed for application in the pharmacokinetics study.（2）To investigate the effects of Fufang Danshen tablets on cytochrome P450 in rats.（3）To establish a high-performance liquid chromatography coupled with diode array detection and evaporative light scattering detection method for simultaneously determining eleven components of three different structural types in Fufang Danshen Tablet.And to assess the quality of Fufang Danshen Tablet basing on Chemometrics.METHODS:（1）The HPLC assay was carried out using a Cosmosil C₁₈column. The mobile phase was acetonitrile-0.05 mol l^-1ammonium acetate buffer with 1% acetic acid（66:34,v/v）.The flow rate was 1.0 ml min^-1.The detection wavelength was set at 263 nm.The plasma concentration of TanshinoneⅠafter bolus injecting was determined using RP-HPLC developed.Moreover,their pharmacokinetics parameters were estimated by DAS1.0.The assay was conducted on a VP-ODS C₁₈column（4.6 min×250 mm;5μm）,and the mobile phase was consisted of methanol and 0.05 mol·L^-1ammonium acetate buffers in a volume ratio of 70:30.The flow rate was 1.0 ml min^-1.The detection wavelength was set at 263 nm.Testosterone propionate was used for internal standard.The plasma concentrations of four tanshinones were determined after oral administration of extraction of Salvia Mitiorrhiza Bunge.And pharmacokinetics parameters were estimated by DAS1.0.（2）The metabolic change of phenacetin being the probe drug was studied in vivo. After given isotonic Na chloride,Cimetidine,phenobarbital sodium and Fufang Danshen tablets by oral administration respectively,the rats were all given phenacetin solution by intra-peritoneal injection.Then the blood samples were collected at different time.An HPLC method developed was used to determine the concentrations of the probe drug in rat plasma.And pharmacokinetics parameters were estimated by DAS1.0.（3）Using the developed HPLC-DAD-ELSD method,five different wavelengths were chosen as the monitoring wavelength to determine salvianolic acid B, dihydrotanshinone,crytotanshinone,tanshinoneⅠ,methylenetanshinone and tanshinoneⅡA,and an evaporative light scattering detector combined was employed to determine five saponins（notoginsenoside R1,ginsenoside Rg1,Re,Rb1 and Rd）.RESULTS:（1）The assay accuracy was better than 92%,and the precision of TanshinoneⅠat low to high concentrations was better than 9 and 11%for intra-day and inter-day assays,respectively.The recovery of the method exceeded 88.3%for TanshinoneⅠ.The assay showed good linearity（r=0.9998）over a relatively wide concentration range from 0.05 to 10.0μg ml^-1.The average concentration-time profiles of TS I after intravenous injection at a dose of 3 mg kg^-1in rats were well described with two-compartment model.After intravenous injection of TS I supernatant solution,TS I was eliminated rapidly with a short value of t_1/2β.The response was linear in the concentration range of 0.01-12.75μg·mL^-1for cryptotanshinone,0.01-16.25μg·mL^-1for tanshinoneⅠ,0.02-13.75μg·mL^-1for methltanshinone,0.02-13.25μg·mL^-1for tanshinoneⅡA,respectively.The recovery of assay was high than 88.7%.The pharmacokinetic paramenters showed that cryptotanshinone and tanshinoneⅡA after oral administration were fitted with two-compartment open models while the methltanshinone and tanshinoneⅠwere fitted with one -compartment open models.（2）The t_1/2of probe drug in groups of isotonic Na chloride,Cimetidine,phenobarbital sodium and Fufang Danshen tablets was 93.51±9.21,161.67±10.95,85.36±8.98, 77.93±6.54 min,respectively.And Fufang Danshen Tablets has certain induction to the rats CYP450.（3）The developed method was successfully applied to quantify the eleven components in fifteen batches Fufang Danshen Tablet samples from four different factories.Moreover,the quality was assessed by principal component analysis and hierarchical clustering analysis;it demonstrated that the preparation procedure and the growth area of raw materials were more important factor influencing the quality of Fufang Danshen Tablet.CONCLUSION:（1）The HPLC method we have developed,was simple, sensitive and specific,and could be used for the analysis of large numbers of plasma samples.The assay was validated to meet the requirements of pharmacokinetic studies, and the results of validation have been showed that this method is sensitive,accurate and reproducible.Therefore,this method could be used to assay multi-components of Radix Salviae Miltiorrhizae in clinical samples and other biological fluid samples following appropriate adjustments.The method developed was simple and reliable and could be used to simultaneously determine the four kinds of tanshinones in rat plasma.（2）Fufang Danshen Tablets has certain induction to the rats CYT450.（3）The results indicated that the method could be used as a convenient and reliable method in the multi-component determination and quality control of traditional Chinese medicine.NEW IDEAS:（1）The pharmacokinetics studies of multi-components in the extraction of Radix Salviae Miltiorrhizae were investigated for the first time.And the developed method was applied to the pharmacokinetic studies of tanshinoneⅠand methltanshinone for the first time.（2）Effects of Fufang Danshen Tablets on liver Cytochrome P450 in Rats was investigated for the first time.（3）A simple quantitative method based on HPLC-DAD-ELSD was developed for the routine analysis of eleven active components in Fufang Danshen Tablet.更多还原