【作者】 刘中博；

【导师】 毕开顺； 陈晓辉； 李军； 王铁杰；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2006， 硕士

【摘要】 本研究以常用中药穿龙薯蓣为研究对象，采用多种色谱分离技术对药材进行了分离纯化，得到了6个单体化合物，通过波谱分析结合理化鉴别，鉴定了它们的化学结构，分别为：薯蓣皂苷元、β-谷甾醇、薯蓣皂苷、苯甲酸、原薯蓣皂苷和延龄草苷。选择高血脂动物模型，以出血、凝血时间，总胆固醇（TC）、甘油三酯（TG）、低密度脂蛋白胆固醇（LDL-C）、高密度脂蛋白胆固醇（HDL-C）的含量，血清超氧化物歧化酶（SOD）、脂质过氧化（LPO）的活性为指标，对其中的4个甾体皂苷进行体外抗自由基、降血脂实验。实验结果显示，4个化合物对降低高脂大鼠血脂水平、降低血液粘滞度和清除机体自由基都有较好作用。其中薯蓣皂苷、原薯蓣皂苷具有较显著的降脂、抗凝、抗自由基作用。本研究建立了测定薯蓣皂苷含量的反相高效液相色谱法。采用Xterra^TM C₁₈柱（250mm×4.6mm i.d.,5μm），流动相为乙腈-水（55：45，v／v），检测波长为203nm。其线性范围为0.1～0.8mg·mL^-1（r=0.9999），方法平均回收率为97.9％（RSD=2.0％）。在同样的色谱条件下，测定不同产地的薯蓣属5种植物中薯蓣皂苷的含量。本方法简便、准确、重复性好，可用于药材、提取物及其制剂的含量测定。本研究首次建立了薯蓣属植物中原薯蓣皂苷的含量测定方法。采用Xterra^TMC₁₈柱（250mm×4.6mm i.d，5μm），流动相为乙腈-水（27：73，v／v），检测波长为203nm。其线性范围为0.02～0.6mg·mL^-1（r=0.9999），方法平均回收率为98.0％（RSD=1.5％）。并且在同样的色谱条件下，测定不同产地的薯蓣属5种植物中原薯蓣皂苷的含量。本方法简便、准确、重复性好，为穿龙薯蓣和其制剂质量评价提供了指标。建立了测定大鼠血浆中原薯蓣皂苷含量的LC-MS分析方法，以延龄草苷为内标，血浆经乙腈沉淀蛋白，离心后提取上清液。原薯蓣皂苷在20～125000ng·mL^-1（r=0.9970）范围内线性关系良好，方法的定量下限为20 ng·mL^-1，日内精密度RSD≤7.8％，日间精密度RSD≤6.8％，准确度RE为±1.8％。研究了大鼠静脉注射0.5，1.0和3.0 mg·kg^-1原薯蓣皂苷后的药物动力学，绘制了原薯蓣皂苷的血药浓度-时间曲线，计算其药物动力学参数。血浆药物浓度-时间曲线下面积(AUC_0-t)随着剂量的增加而增加，二者呈良好的线性关系：y=1.388×10³x+28.81，r=0.997；排泄速率常数随着剂量的增加而增加，而清除率和稳态分布容积则随着剂量的增加而下降。表明药物在大鼠体内可能存在剂量相关性药动学特征。更多还原

【Abstract】 Dioscorea nipponica Mak. is a native perennial herb distributed throughout China. Its rhizomes have been known in traditional Chinese medicine （TCM ） as Chuanlongshuyu,which used for the treatment of gastropathy,anthrax,rheumatic heart disease and rheumarthritis. Chuanlongshuyu is one of the important folk medicines in TCM.In order to isolate the active constituents in Chuanlongshuyu, various chromatographic techniques were performed. Six compounds were isolated and identified with IR, NMR and MS data and physical-chemical properties. They were diogenhuβ-itosterol、dioscin、benzoic acid、protodioscin、trillin. The investigation on reducing blood liquids and free radical effects of these compounds were investigated （in vivo）. It was suggested that four steroid sa ponins possessed reducing activity to blood liquids and free radical.Dioscin has been reporting as one of the effective components. We established a method for the determination of dioscin in Chuanlongshuyu and Dioscorea by RP-HPLC. The separation was achieved on a Xterra^? Cis column（250 mm×4.6 mm, 5μm）using acetonitrile-water （55:45） as the mobile phase at a flow rate of 1.0 mL·min^-1, 203 nm as the detection wavelength. Dioscin was well-separated. The linear range of dioscin was 0.1～0.8 mg·mL^-1 （r = 0.9999）. The average recovery was 97.9% with RSD of 2.0% （n = 9） .The assay was found to be simple and accurate to measure the concentrations of dioscin in Chuanlongshuyu and its preparation.To establish a method for the determination of protodioscin in Chuanlongshuyu by RP-HPLC . The chromatographic condition included a Xterra^? C₁₈ column （250 mm×4.6 mm i.d, 5μm） and the mobile phase consisting of acetonitrile-water （27:73, v/v） and the absorbance was monitored at 203 nm. The calibration curve was linear over the range of 0.02～0.6 mg·mL^-1 for protodioscin （r=0.9999, n=6）. The average recovery was 98.0% （RSD=1.5%）. The assay was found to be simple and accurate to measure the concentrations of protodioscin in Chuanlongshuyu and its preparation.We have established a rapid, sensitive and selective LC-ESI-MS/MS method to measure protodioscin in rat plasma and investigated the pharmacokinetics of protodioscin after intravenous administrations. Plasma samples （40μL） were prepared after plasma protein precipitation, and a 20μL aliquot of the supernatant was injected directly onto a analytical column （50 mm×2.0 mm i.d.） with an mobile phase consisted of acetonitrile-water-formic acid （80:20:0.1, v/v/v）. Analytes were detected with a LC-ESI-MS/MS system in positive multiple reaction monitoring mode (m/z 1032.6 （precursor ion） to m/z 869.7 （product ion） for protodioscin; m/z 577.1 （precursor ion） to m/z 253.1 （product ion） for trillin（internal standard）. The sensitivity of the method was 20 ng/mL and a linear range of 20-125000 ng/mL. The intra-and inter-day relative standard deviation （RSD） across three validation runs over the entire concentration range was＜8.0%. Accuracy determined at three concentrations （50, 5000 and 50000 ng/mL for protodioscin） ranged from 0.2 to 1.8% as terms of relative error （R.E.）. Each plasma sample was chromatographed within 3.5 min. This LC-ESI-MS/MS method allows accurate, high-throughput analysis of protodioscin in small amounts of plasma and possibly other biological samples.更多还原