【作者】 李德强；

【导师】 张兰桐；

【作者基本信息】 河北医科大学 ， 药物分析学， 2008， 硕士

【摘要】 中药金莲花为毛茛科植物金莲花（Trollius chinensis Bunge.）的干燥花,在我国民间作为药用植物由来已久。《本草纲目拾遗》中谓其“味苦,性寒,无毒”,可“治口疮,喉肿,浮热牙宣,耳疼,目痛”,具有“明目,解岚瘴”的功效。现代药理研究表明,金莲花具有抗菌、抗病毒等活性,广泛用于治疗感冒、发烧、慢性扁桃体炎、急性鼓膜炎、尿路感染和其他炎症。据文献报道,金莲花主要含有黄酮、有机酸等成分,水提物具有较强的抗病毒活性,其主要活性成分为黄酮类化合物。中药药物动力学是在中医理论指导下,利用动力学原理和数学处理方法,定量地描述中药有效成分、有效部位、中药单方与复方制剂通过各种给药途径进入体内后的吸收、分布、代谢和排泄等一系列过程的动态变化规律及其体内时量?时效关系,是中药现代化研究的重要内容之一,可以使传统天然药物化学研究与药理药效研究有机地结合起来,对阐明中药的有效性、探求中药的药效物质基础和作用机制等都具有重要意义。本论文对金莲花进行了提取分离,制备了荭草素-2′′-O-β-L-半乳糖苷、荭草苷和牡荆苷三种单体成分,分别探讨了大鼠静注金莲花提取物后,主要有效成分荭草素-2′′-O-β-L-半乳糖苷、荭草苷和牡荆苷的动力学特点和大鼠静注荭草苷单体后在其体内动力学、组织分布特征,为阐明金莲花的作用机制和药效物质基础研究做出了有意义的探索。第一部分金莲花化学成分研究（荭草素-2′′-O-β-L-半乳糖苷、荭草苷和牡荆苷对照品的制备）目的:从金莲花中分离制备荭草素-2′′-O-β-L-半乳糖苷、荭草苷和牡荆苷三种单体成分。方法:结合硅胶柱层析、制备HPLC等方法对金莲花醇提取物进行系统分离纯化,通过薄层、UV、HPLC等方法对其分离产物进行鉴定。结果:提取分离得到三个化合物,分别鉴定为荭草素-2′′-O-β-L-半乳糖苷、荭草苷和牡荆苷。所得荭草素-2′′-O-β-L-半乳糖苷、荭草苷和牡荆苷用归一化法定量,纯度均大于98%。结论:本法简便,制备成本低,荭草素-2′′-O-β-L-半乳糖苷、荭草苷和牡荆苷可作为定性定量分析使用的对照品,同时可用于药物代谢动力学的研究。第二部分金莲花提取物中三种黄酮苷的药动学研究目的:建立高效液相色谱法（HPLC）同时测定大鼠血浆中荭草素-2′′-O-β-L-半乳糖苷、荭草苷和牡荆苷浓度的方法,并将其用于SD大鼠静注金莲花提取物后血浆中三种黄酮苷的药动学研究。方法:大鼠以4mL·kg-1静注给予提取物溶液,分别于给药后2,5,10,15,20,30,40,50,60,90,120和180min眼眶静脉取血,置于肝素化塑料离心管中,血浆样品采用甲醇沉淀蛋白的方法;色谱柱为C18柱,流动相为乙腈-0.1%醋酸水,梯度洗脱（0¹2min,18²0%乙腈;12.5min,18%乙腈）,流速为1mL·min-1,检测波长348nm。结果:荭草素-2′′-O-β-L-半乳糖苷、荭草苷和牡荆苷的线性范围分别为0.315¹61,0.326¹67和0.215¹10μg·mL^-1,最低检测限分别为0.04,0.03和0.03μg·mL^-1。日内和日间精密度（RSD）在1.68%和8.43%之间,准确度在（RE）8.55%之下。其提取回收率在70.35%和86.42%之间。大鼠单次静注金莲花提取液4mL·kg-1后,三个主要药效成分荭草素-2′′-O-β-L-半乳糖苷、荭草苷和牡荆苷的的动力学参数如下:t1/2α为1.72±0.58,1.88±0.23和1.22±0.42min,t1/2β为10.38±5.75,11.88±0.46和9.99±1.05min,t1/2γ为36.88±3.71,64.50±9.22和45.64±7.66min,V（c）为0.018±0.007,0.016±0.001和0.015±0.006L·kg-1,CL（S）为0.002±0.000, 0.002±0.000和0.003±0.000L（·kg·min）-1,AUC（0-t）为3539.4±103.9,2840.8±215.6和581.5±59.5mg·min·L^-1,MRT（0-t）为31.46±0.88,15.86±0.59和14.25±0.48min。结论:本章建立了同时测定血浆中荭草素-2′′-O-β-L-半乳糖苷,荭草苷,牡荆苷的高效液相色谱法,并将其应用于SD大鼠静注金莲花提取物后3种黄酮苷的药动学研究。该方法具有较高的灵敏度和专属性,经方法学验证,方法具有良好的精密度和准确度,符合生物样品分析的要求。第三部分荭草苷在大鼠体内的药动学和组织分布研究目的:1.建立高效液相色谱法（HPLC）测定大鼠血浆中荭草苷浓度的方法,并研究不同剂量单次给药后荭草苷单体在大鼠体内的药物动力学过程。2.研究荭草苷在大鼠体内的组织分布。方法:1.大鼠以静注给予荭草苷溶液,分别于给药后2,4,8,12,16,20,25,30,45,60和90min眼眶静脉取血,置于肝素化塑料离心管中,样品预处理采用甲醇沉淀蛋白后氮气吹干,流动相复溶,利用HPLC内标法（葛根素）,色谱柱为反相C18柱（250mm×4.6mm,5μm）,流动相为乙腈-0.1%醋酸水（20:80）,流速为1mL·min-1,检测波长250nm用于检测内标物葛根素,348nm用于检测荭草苷;2.取大鼠随机分为6组,静注给予20mg·kg-1荭草苷后,于5,15,30,45,60和90min分别放血处死,取心、肝、肺、脾、肾、脑、胃和肠,经生理盐水冲洗,去除少量的淤血或组织内容物,并用滤纸吸去水分,称重后于匀浆器中制成500mg·mL^-1的生理盐水匀浆,采用甲醇沉淀蛋白后氮气吹干流动相复溶的方法,利用HPLC法（除没用内标外同上方法1）,测定组织中的荭草苷含量。结果:1.荭草苷的线性范围0.250⁵0.1μg·mL^-1,最低定量限为0.250μg·mL^-1,RE为–2.62%,RSD为3.2%（n=6）,0.10, 5.00和25.00μg·mL^-1三种浓度的日内、日间精密度（RSD）分别为3.0%⁴.5%和2.9%⁶.5%,准确度（RE）为0.87%⁵.6%,回收率68.0%⁷3.1%,稳定性研究结果表明样品稳定。大鼠单次按三种不同剂量10、20和30mg·kg-1分别静注荭草苷后,主要药动学参数分别如下:V（c）为0.364±0.085,0.233±0.014和0.259±0.023L·kg-1,CL（S）为0.057±0.022,0.035±0.020和0.049±0.003L·（kg·min）-1,T1/2γ为14.34±2.16,22.53±3.48和25.29±3.27min,AUC0-t为150.601±11.528,422.392±54.063和593.473±25.516μg·min·mL^-1。2.高、中、低三种浓度的日内、日间精密度均小于15%,回收率均大于65%。大鼠尾静脉注射荭草苷20mg·kg-1后,肝、肺和肾中药物浓度最高,在胃、心和脾等组织中浓度较低,脑组织中药物浓度极低。结论:1.荭草苷静注后在大鼠体内消除较快,在10³0mg·kg-1范围内呈线性动力学;2.荭草苷主要分布在肝,肾和肺等组织,难以通过血脑屏障进入脑组织;在所研究的组织中,未见明显的特殊蓄积现象。更多还原

【Abstract】 Jinlianhua, dried flowers of Trollius chinensis Bunge, has been used as an important traditional Chinese medicine （TCM） in Chinese folk for a long history.《Bencao Gangmu Shiyi》said that it was“bitter in taste, cold-natured,asepsis”,and could be used for the treatment of aphtha,larynx boss,light fever atrophy of the gum,ear ache,ophthalmalgia”,and that it had the effects of eyesight improvement and anti-miasma. Modern pharmacological studies suggest that it possesses antimicrobial and antiviral actions and has been used widely to treat cold, fever, chronic tonsillitis,acute tympanitis,urinary tract infection and other inflammations. Previous studies demonstrated that trollius mianly contains flavonoids and organic acid components, the aqueous extract of which has a strong antiviral action, and its main active compenents are flavonoids.With the help of dynamic and kinetic mechanisms and mathematics method, pharmacokinetics of traditional Chinese medicine （TCM）,under the guide of chinese medical science theory, studied absorption, distribution, biotransformation and elimination of active constituents or parts, simple or compound recipes of TCM, and relations of pharmacokinetics and pharmacodynamics of TCM. As one of important contents of modernization of TCM, pharmacokinetics of TCM might be combine the studies of phytochemistry and pharmacodynamics and were beneficial for illuminating efficacy of TCM, identifying therapeutical basis and mechanism of action of TCM.In the present study, a separation method of orientin-2”-O -β-L-galactopyranosyl（OGA）, orientin and vitexin from Trollius chinensis was established. Pharmcokinetic of OGA, orientin and vitexin after intravonous administration of Trollius chinensis extract （TCE） and pharmcokinetic and tissue distribution of orientin after intravonous administration of oreintin monomer to rats were investigated for illuminating mechanism of action of theirs. The research provided a significant exploration for pharmacokinetics and therapeutic basis of TCM.Part 1 Study on chemical constituents of Trollius chinensis （Preparation of orientin-2′′-O-β-L-galactopyranosyl, orientin and vitexin）Objective: To establish a separation method of orientin-2′′-O-β-L-galactopyranosyl, orientin and vitexin from Trollius chinensi.Methods: After extracted with alcohol and precipitated with hot water from Trollius chinensi, the extract was isolated and purified by silica gel column chromatography and preparative HPLC. Orientin-2′′-O-β-L-galactopyranosyl, orientin and vitexin were identified by thin-layer chromatography （TLC） and high performance liquid chromatography （HPLC）.Results: Three compounds were separated from Trollius chinensi and identified as: Orientin-2′′-O-β-L-galactopyranosyl, orientin and vitexin. The purity of orientin-2′′-O-β-L-galactopyr -anosyl, orientin and vitexin than 98% by normalization method of HPLC.Conclusion: The developed method is simple and at low production cost. The products can be used as reference substance for quality control and drugs for pharmacokinetic study.Part 2 Studies on pharmacokinetics of three active flavonoids in rats after intravenous administration of Trollius chinensis extractObjective: To develople a HPLC method for simultaneous determination of orientin-2′′-O-β-L-galactopyranosyl, orientin and vitexin in rat plasma for studying the pharmacokinetics of them after a single intravenous administration of Trollius chinensis extract.Methods: After rats were intravenous administrated extract solution at a dose of 4mL·kg^-1, blood samples were obtained from fossa orbitalis vein according to the specific schedule, 2, 5, 10, 15, 20, 30, 40, 50, 60, 90, 120 and 180min and collected in heparinized centrifuge tube, respectively. Sample was pretreated by a single-step protein precipitation with methanol. The RP-C18 column （250×4.6mm,5μm）was used as the stationary phase with the gradient mobile phase consisting of methanol and 0.1% acetic water. The flow rate was 1mL·min-1, the UV detector was set at 348nm.Results: Calibration curves of orientin-2′′-O-β-L-galactop -yranosyl, orientin and vitexin were generated over the range 0.315¹61, 0.326¹67 and 0.215¹10μg/mL, respectively, and the LOQ of them were 0.04, 0.03 and 0.03μg/mL, respectively. The intra- and inter-day precisions （RSD） for the analysis of three analytes were between 1.68% and 8.43% with accuracies （RE） below 8.55%. The mean extraction recoveries were between 70.35% and 86.42%. After single intravenous administration of 4mL·kg^-1 extract solution of Trollius chinensis to rats, the main pharmacokinetic parameters of orientin-2′′- O-β-L-galactopyranosyl, orientin and vitexin were as follows: t1/2αwere 1.72±0.58, 1.88±0.23 and 1.22±0.42min, t1/2βwere 10.38±5.75, 11.88±0.46 and 9.99±1.05min,t1/2γwere 36.88±3.71, 64.50±9.22 and 45.64±7.66min,V（c）were 0.018±0.007, 0.016±0.001 and 0.015±0.006L·kg^-1, CL（S）were 0.002±0.000, 0.002±0.000 and 0.003±0.000L·(kg·min-1,AUC（0-t）were 3539.4±103.9, 2840.8±215.6 and 581.5±59.5mg·min·L^-1,MRT（0-t）were 31.46±0.88, 15.86±0.59 and 14.25±0.48min。Conclusion: A simple HPLC assay method has been developed for simultaneous determination of orientin-2′′-O-β-L- galactopyranosyl, orientin and vitexin in rat plasma following an intravenous administration of the extract of Trollius chinensis and the method was was successfully applied to the pharmacokinetic studies of the three active components. The method with high sensitivity, specificity, good precision and accuracy is fit to the needs of biosample analysis.Part 3 Studies on pharmacokinetic and tissue distribution of orientin in ratsObjective: 1. A HPLC method for determination of orientin in rat plasma was developed for studying the pharmacokinetics of orientin in rats after a single intravenous administration of orientin at three different dosages. 2. A HPLC method was developed and validated for determination of orientin in rat tissues.Methods: 1. After rats were intravenous administrated orientin, blood samples were obtained from fossa orbitalis vein according to the specific schedule, 2,4,8,12,16,20,25,30,45,60 and 90min and collected in heparinized centrifuge tube, respectively. Sample was pretreated by protein precipitation with methanol using puerarin as internal standar （IS）,the supernatant was collected and evaporated to dryness at 40℃under a gentle stream of nitrogen. The residue was then reconstituted with 100μL mobile phase, and centrifuged at 12000×g for 5 min, and an aliquot （20μL） of the supernatant was injected into the HPLC system. The RP-C18 column （250×4.6mm,5μm） was used as the stationary phase with the mobile phase consisting of acetonitrile–0.1% acetic water （20:80, v/v） at a flow rate of 1.0mL·min-1. Chromatograms were monitored at 250nm for IS, 348nm for orientin and the temperature of column was kept at 35℃. The flow rate was 1mL·min-1. 2. Rats were randomly assigned to six groups. After intravenous administration of 20mg·kg^-1 solution of orientin, heart, liver, lung, spleen, kidney, brain, stomach and small intestine samples were obtained at 5, 15, 30, 45, 60 and 90min, respectively. Tissue samples were weighed rapidly and put into normal saline solution to remove the blood or content, blotted on filter paper, and then were weighed for wet weight and homogenized in saline solution (500mg·mL^-1). Preparation of tissues samples and HPLC analysis conditon were same with the plasma samples, except that IS was not used. The method was applied to study tissues distribution of oreintin in rats after a single administration of orientin at a dose of 20mg·kg^-1.Results: 1. The calibration curve in plasma was linear over the range of 0.250⁵0.1μg·mL^-1 and the RSD values of intra-day and inter-day were less than 15%. The recovery of orientin in rat plasma was 68.0%⁷3.1%. The study of stability demonstrated samples were stable. After single intravenous of three different dosages 10, 20 and 30mg·kg^-1 orientin to rats, the main pharmacokinetic parameters were as follows: V（c） were 0.364±0.085, 0.233±0.014 and 0.259±0.023L·kg^-1, CL（S） were 0.057±0.022, 0.035±0.020 and 0.049±0.003L·（kg·min）-1, T1/2γwere 14.34±2.16, 22.53±3.48 and 25.29±3.27min,AUC0-t were 150.601±11.528, 422.392±54.063 and 593.473±25.516μg·min·mL^-1. 2. The intra-day and inter-day presion were less than 15%, and the extraction recoveries of different tissues were larger than 65%. After a single administration of orietin at a dose of 20mg·kg^-1, the highest level was observed in kidney, then in liver and lung. The lowest level was found in brain.Conclusion: 1. Orientin was eliminated rapidly in rat and manifested linear dynamics at 10³0mg·kg^-1 range. 2. Kidney、liver and lung were the main distribution tissues of orientin in rats, and orientin had difficulties in crossing the blood brain barrier. It was also found there was no accumulation of loganin in rat tissues.更多还原