【作者】 李清；

【导师】 毕开顺；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2006， 博士

【摘要】 本研究以部颁抗癌中药艾迪注射液为研究对象，对其制剂工艺、药效学、质量标准和有关成分药动学进行了初步研究，内容如下：采用正交试验设计方法，对注射用艾迪（冻干）提取工艺进行优化。分别以斑蝥素、人参皂苷Rg₁与Re总含量、异秦皮啶为考察指标，对提取溶剂用量、提取时间和提取次数3个因素进行优化，确定了注射用艾迪（冻干）中各组成药味的最佳提取工艺。采用D-101大孔树脂分离、超滤等方法对制剂进行精制，同时确定了真空冷冻干燥条件。结果表明，制成的冻干粉针呈浅黄色疏松状固体，含水量低，加水后能迅速溶解且分散均匀。采用MTT法，选取体外培养的人肝癌Bel-7402、人宫颈癌HeLa、人黑色素瘤A375-S2、人胃腺癌SGC-7901和小鼠纤维肉瘤L929细胞为模型，以半数抑制浓度(IC₅₀)为指标，考察了注射用艾迪（冻干）对上述肿瘤细胞的生长抑制作用，结果表明，该制剂对上述肿瘤细胞有显著的增殖抑制作用，IC₅₀分别为0.0162g·mL^-1、0.0796g·mL^-1、0.0982g·mL^-1、0.0119g·mL^-1和0.0144g·mL^-1（生药量）。以小鼠移植性肿瘤肝癌H22、肉瘤S180和C57BL/6小鼠Lewis肺癌荷瘤小鼠为模型，抑瘤率为指标，考察了注射用艾迪（冻干）的体内抗肿瘤作用，结果表明，该制剂在三种不同剂量下对小鼠H22肝癌、S180肉瘤和C57BL/6小鼠Lewis肺癌均有抑制作用，并且抑制作用呈现明显的剂量依赖性。采用薄层色谱法分别对注射用艾迪（冻干）中的斑蝥素、人参皂苷Rg₁、人参皂苷Rb₁、人参皂苷Re、黄芪甲苷和异秦皮啶进行定性鉴别，斑点清晰，重复性好，专属性强，可用于该制剂中各组成药味的鉴别。建立了该制剂的GC和HPLC指纹图谱质量评价方法。采用DB-1石英毛细管气相色谱柱（30m×0.25mm×0.25μm），以120℃保持5min，3℃·min^-1升至250℃，再以2℃·min^-1升至280℃保持30min为升温程序，以斑蝥素为参照物，建立了GC指纹图谱鉴别方法；采用Agilent Zorbax SB-C₁₈液相色谱柱（250mm×4.6mm，5μm），以乙腈-0.05％磷酸水溶液为流动相进行梯度洗脱，以紫丁香苷为参照物，建立了HPLC指纹图谱鉴别方法。结果表明该方法能有效控制该制剂与中间体的质量。采用GC-FID法测定了注射用艾迪（冻干）中斑蝥素含量。选用DB-1毛细管柱，柱温为145℃，载气为N₂，斑蝥素在2.46～49.20μg·mL^-1浓度范围内线性关系良好，平均回收率为100.3％（RSD=2.8％）；采用HPLC-UV法同时测定了人参皂苷Rg1和Re含量。选用Hypersil C₁₈色谱柱，柱温为35℃，流动相为乙腈-0.05％磷酸溶液（19∶81），二者分别在20～200μg·mL^-1（r=0.9997）和10～100μg’mL^-1（r=0.9996）浓度范围内线性关系良好，平均回收率分别为98.2％（RSD=1.1％）和97.2％（RSD=1.2％）；采用HPLC-DAD法同时测定了刺五加药材中的原儿查酸、紫丁香苷B、绿原酸、咖啡酸、紫丁香苷E和异秦皮啶含量，选用Agilent Zorbax SB-C₁₈色谱柱，柱温为35℃，以乙腈-0.05％磷酸水溶液为流动相进行梯度洗脱，各组分线性关系良好，平均回收率为90.3～94.3％，RSD＜2.4％。采用相同色谱条件，同时测定了制剂中毛蕊异黄酮-7-O-β-D-葡萄糖苷、紫丁香苷B、绿原酸、咖啡酸、紫丁香苷E和异秦皮啶的含量，各组分线性关系良好，平均回收率为96.6～98.2％，RSD＜2.3％。所建立的含量测定方法为有效控制该制剂质量提供了定量依据。建立了测定大鼠血浆中异秦皮啶含量的HPLC-UV分析方法，以香豆素为内标，乙酸乙酯为提取溶剂，异秦皮啶在0.05～8.0μg·mL^-1范围内线性关系良好。方法的定量下限0.05μg·mL^-1，日内精密度RSD≤6.4％，日间精密度RSD≤7.9％，准确度RE为±5.1％，平均回收率为85.4％。测定了异秦皮啶的血药浓度-时间曲线，并计算了相应的药物动力学参数，t_1/2为4.26h，C_max为5.47μg·mL^-1，t_max为0.24h。建立了同时测定大鼠血浆中紫丁香苷B和绿原酸含量的HPLC-DAD分析方法，以栀子苷为内标，血浆经甲醇沉淀蛋白后，采用选择波长法进行分析。紫丁香苷B和绿原酸分别在0.20～10μg·mL^-1和0.25～30μg·mL^-1范围内线性关系良好。定量下限分别为0.20μg·mL^-1和0.25μg·mL^-1，日内精密度分别为RSD≤8.5％和RSD≤6.1％，日间精密度分别为RSD≤7.1％和RSD≤5.5％，准确度RE分别为±4.1％和±8.6％，平均回收率分别为92.1％和80.9％。静脉给予注射用艾迪（冻干）后，大鼠血浆中紫丁香苷B消除较快，t_1/2为0.49h；绿原酸的t_1/2为0.82h。建立了测定大鼠血浆中毛蕊异黄酮-7-O-β-D-葡萄糖苷含量的HPLC-UV分析方法，以香兰素为内标，乙酸乙酯为提取溶剂，毛蕊异黄酮-7-O-β-D-葡萄糖苷在0.11～17.6μg·mL^-1范围内线性关系良好。方法的定量下限为0.11μg·mL^-1，日内精密度RSD≤6.3％，日间精密度RSD≤6.2％，准确度RE为±6.7％，平均回收率为87.2％。测定了毛蕊异黄酮-7-O-β-D-葡萄糖苷的血药浓度-时间曲线，并计算了相应的药物动力学参数，t_1/2为0.70h，AUC_0-t、AUC_0-∞分别为1.38μg·h·mL^-1和1.64μg·h·mL^-1。更多还原

【Abstract】 Aidi liquid injection was a clinical medicine commonly used in China for thetreatment of cancer, which was listed in ministerial standard. In this paperpreparation procedure, anti-tumor activity, quality control method andpharmacokinetics on a few active constitutes of Aidi lyophilizer were studied.The preparation procedure of Aidi lyophilizer was established using differentkinds of methods and techniques. The optimal extraction process of Aidilyophilizer was studied by orthogonal design and the contents of cantharidin,ginsenoside Rg₁, ginsenoside Re and isofraxidin as quality assessment indices.Three factors were studied in this experiment, including the solvent consumption,duration of extraction and times of extraction. D-101 macroporous resin and ultrafiltration were selected to optimize the refining process and lyophilization was alsostudied. The lyophilizer was yellowish freeze-dried powder and solved easily inwater.The anti-tumor activity of Aidi lyophilizer in vitro and vivo were studied. Theantiproliferative activities of Aidi lyophilizer against human hepatocarcinoma cellline BEL-7402, human cervix epithelial cell line HeLa, human malignantmelanoma cell line A375-S2, human gastric carcinoma cell line SGC-7901 andmurine fibrosarcoma cell line L929 were evaluated by MTT colorimetric assay invitro. The results showed Aidi lyophilizer possessed the antiproliferative activityagainst different tumor lines. IC₅₀ were 0.0162, 0.0796, 0.0982, 0.0119 and0.0144g·mL^-1, respectively. The inhibitory effects of Aidi lyophilizer on tumorgrowth were observed by the models of implanted sarcoma 180 （S180） andhepatoma 22 （H22） in mice and Lewis lung-cancer in C57BL/6 mice. The resultsshowed Aidi lyophilizer restrained the growth of tumor in mice in adose-dependent manner.TLC methods were developed for the identification of cantharidin in Mylabris, ginsenoside Rg₁ and ginsenoside Re in Radix Ginseng, astragaloside in RadixAstragali and isofraxidin in Radix Acanthopanacis. GC fingerprint was carried outon a DB-1 column with cantharidin as internal reference. Column temperaturemaintained 10min at 120℃, then raised to 250℃with rate of 3℃·min^-1, and thenraised to 280℃with rate of 2℃·min^-1, and maintained 30min at 280℃. HPLCfingerprint was separated on a Agilent Zorbax SB-C₁₈ column with syringin asinternal reference. GC and HPLC fingerprints were applied to analyze quality ofAidi lyophilizer and its intermediate.The determination of cantharidin was carried out by using GC-FID method,which showed a good linearity from 2.46 to 49.2μg·mL^-1 with a mean recovery of100.3%. The acetonitrile-water containing 0.05% phosphate acid （19:81） solutionwas adopted as the mobile phase for the determination of ginsenoside Rg₁ and Reon an Hypersil C₁₈ column. It showed good linearities from 20 to 200μg·mL^-1 forginsenoside Rg₁ with a mean recovery.of 98.2% and from 10 to 100μg·mL^-1 forginsenoside Re with a mean recovery of 97.2%. A ttPLC-DAD method wasdeveloped for simultaneous determination of .protocatechuic acid, syringin,chlorogenic acid, caffeic acid, liriodendrin and isofraxidin in RadixAcanthopanacis, collected from different batches. The determination wasaccomplished by a gradient elution system. The linear calibration curves wereplotted with satisfied relative coefficients. The average recoveries were in therange of 90.3～94.3 % with RSD less than 2.4 %. With the same gradient elutionsystem, calycosin-7-O-beta-D-glucoside, syringin, chlorogenic acid, caffeic acid,liriodendrin and isofraxidin were determined in Aidi lyophilizer. The linearcalibration curves were plotted with satisfied relative coefficients. The averagerecoveries were in the range of 96.6～98.2 % with RSD less than 2.3 %.A high performance liquid chromatography （HPLC） method was developed toquantify isofraxidin in rat plasma using liquid-liquid extraction and coumarin asinternal standard. The linear range was 0.05～8.0μg·mL^-1 and the low quantificationlimit was 0.05μg·mL^-1. The intra- and inter-day relative standard deviations （RSD）in the measurement of quality control （QC） samples 0.25, 2.0 and 6.0μg·mL^-1ranged from 5.7% to 6.4% and 6.3% to 7.9%, respectively. The accuracy was±5.1% in terms of relative error （RE）. The mean extraction recovery was 84.9%. The corresponding pharmacokinetic parameters were obtained, t_1/2 was 4.26h, C_maxwas 5.47μg·mL^-1 and t_max was 0.24h.A simple and sensitive high-performance liquid chromatographic method wasdeveloped to simultaneously quantify syringin and chlorogenic acid in rat plasmausing wavelength-transfer technology. After deproteinized by methanol, the plasmasamples were analyzed with gardenoside as internal standards. The linear rangeswere 0.20～10μg·mL^-1 and 0.25～30μg·mL^-1, respectively. The lower limits ofquantification were 0.20μg·mL^-1 and 0.25μg·mL^-1, respectively. The method wasshown to be reproducible and reliable with intraday precision below 8.5% and6.1%, inter-day precision below 7.1% and 5.5%, accuracy within±7.1% and±8.6%,and mean extraction recovery excess of 92.1% and 80.9%, respectively, whichwere all calculated from the blank plasma sample spiked with syringin andchlorogenic acid at three concentrations of 0.20, 1.0 and 6.0μg·mL^-1 for syringinand 0.25, 2.0 and 20μg·mL^-1 for chlorogenic acid. Since the concentration ofsyringin in plasma samples was lower, it was determined only 1.5h afterintravenous administration of Aidi lyophilizer, t_1/2 of syringin and chlorogenic acidwere 0.49h and 0.82h, respectively.A specificity and accuracy high performance liquid chromatographic methodwas developed to quantify calycosin-7-O-beta-D-glucoside in rat plasma usingliquid-liquid extraction and vanillin as internal standard. The linear range was0.11～17.6μg·mL^-1 and the low quantification limit was 0.11μg·mL^-1. The intra-andinter-day relative standard deviations （RSD）in the measurement of quality control（QC） samples 0.11, 0.22, 1.32 and 8.80μg·mL^-1 ranged from 4.1% to 6.3% and4.3% to 6.2%, respectively. The accuracy was from -6.7% to 4.3% in terms ofrelative error （RE）. The validated method was used to take a limited view of thepharmacokinetic profile of calycosin-7-O-beta-D-glucoside in rat plasma aftertaken Aidi lyophilizer. The main pharmacokinetic parameters were:AUC_0-t=1.38μg·h·mL^-1, AUC_0-∞=1.64μg·h·mL^-1 and t_1/2=0.70h, respectively.更多还原