hplc法测定龙芪柔肝胶囊中丹参酮‖a的含量 中药学专业毕业论文

毕业论文HPLC法测定龙芪柔肝胶囊中丹参酮A的含量系别生物技术系班级中药制药2班姓名学号指导老师目录摘要1前言11龙芪柔肝胶囊简介（3）12处方分析（3）13中药药理学分析（3）14小结（3）2正文21仪器与试药（3）211仪器3212试药322方法与结果3221对照品溶液的制备3222供试品溶液的制备4223色谱条件4224线性关系的考察4225精密度试验4226重复性试验4227稳定性试验4228加样回收率试验4229样品的含量测定（4）3讨论31检测波长的选择（4）32色谱条件的选择（4）33提取方法的选择（4）331药品预处理（5）332供试品溶液的提取（5）333供试品的筛选（5）4参考文献（5）5致谢（5）HPLC法测定龙芪柔肝胶囊中丹参酮A的含量摘要目的建立HPLC法测定龙芪柔肝胶囊中丹参酮A含量的方法。方法选用ANJILENTC18柱（250MM46MM,5UM）,以甲醇水（2080）为流动相，流速10MLMIN1,检测波长270NM,柱温25。结果丹参酮IIA在169615264UGML间呈良好的线性关系R2099969,平均加样回收率为10132（RSD28N5）；结论该方法快速、准确、精密，可用于龙芪柔肝胶囊的质量控制。关键字HPLC法；龙芪柔肝胶囊；丹参酮IIADETERMINATIONOFTANSHINONETONGAINTHELONGQICAPSULESBYHPLCABSTRACTOBJECTIVETOESTABLISHANHPLCMETHODFORTHETANSHINONETONGAINTHELONGQICAPSULESMETHODTHEANALYSISWASPERFORMEDONACOLUMNOFANJILENTC18（250MM46MM,5UM）THEMOBILEPHASECONSISTEDOFMETHANOLANDWATER（2080）WITHTHEFLOWRATEOF10MLMIN1THEDETECTIONWAVELENGTHWAS270NMANDTHECOLUMNTEMPERATUREWAS25RESULTPROMISINGLINEARITYWASOBTAINEDWITHINTHERANGEOF169615264UGMLFORTANSHINONETONGATHEAVERAGERECOVERYOFTANSHINONETONGAWASR2099969CONCLUSIONTHISMETHODISRAPIDANDACCURATEANDCANBEUSEDTOCONTROLTHEQUALITYOFTANSHINONETONGAKEYWORDSHPLCTANSHINONETONGALONGQICAPSULES1前言11龙芪柔肝胶囊简介龙芪柔肝胶囊为信阳市中医院院内制剂，由黄芪、丹参、党参、生地黄、北沙参、当归、地龙、醋白芍、鳖甲、黄精、五味子、灵芝、桃仁、红花、炒穿山甲共15位中药制备而成。主治慢性病毒性肝炎，肝纤维化、肝硬化等症。具有益气养阴，活血柔肝的功效。方中地龙有较强的异味，穿山甲属名贵药材以打粉入药。因胶囊剂具有能够掩盖药物不良臭味，载药量大的特点，故选用胶囊剂4。12处方分析方中以黄芪、地龙为君药，以丹参、当归、生地黄、北沙参、党参为臣药。方中黄芪甘温入脾肺经，补中气、升清阳，益肺气、实皮毛，以扶正；地龙同入肝脾经，息风止痉，通络，以开血脉阻滞，二者共为君药。丹参、当归并用，生血、活血且化瘀、通络而不伤血；生地、北沙参并用，滋阴而又养阴；党参补气以行血；五者共为臣药。取君二臣五，奇之制之意。五味子、醋白芍共同辅助君药抑阳敛阴，以柔肝；鳖甲、黄精、灵芝共用助君药滋补肝阴。五者共为佐药。桃仁、红花活血祛瘀，穿山甲消肿排脓，三者共同引药归经，均为使药。本方为补中益气汤（脾胃论）和补阳还五汤（医林改错）的复合方。一为补气，一为活血祛瘀，二者并用以达到益气养阴、活血柔肝的功效5。13中药药理学分析黄芪主含多糖类和皂苷类成分，能增强非特异性免疫功能，包括增加WBC和中性粒细胞、M吞噬功能。地龙提取物口服，对小鼠S180和H22均有抑瘤作用，并可增强5FU受体活化，从而增强免疫细胞的活性。丹参中主含丹参酮A，丹参酮A磺酸钠可抑制心肌收缩力，降低窦房结自律性，能促进肝、骨、皮肤等多种组织的修复与再生，其中促进肝组织的修复与再生尤为显著3。因此丹参酮A也是本药的重要和关键性成份。14小结本文重点探索了HPLC法测定龙芪柔肝胶囊中丹参酮A含量时方法的建立及优化，为全面评价该制剂质量和完善本复方制剂的质量提供实验依据。2正文21仪器与试药211仪器AGILENT1200液相色谱仪美国AGILENT公司，AGILENT1200检测器，AGILENT工作站；WATERS1525液相色谱仪（WATERS公司，WATERS2489柱温箱BREEZE工作站）；普利赛斯双量程电子天平美国塞利普斯公司；超纯水机（基因型185OD重庆摩尔制水设备有限公司）；超声仪（宁波新芝生物科技有限公司）。212试药丹参酮A对照品（规格20MG支，110766200518，中国药品生物制品检定所）；甲醇（色谱纯，美国AGILENT公司）；龙芪柔肝胶囊（信阳市中医院制剂室，规格60粒瓶，批号20110410、20110512、20110823）；水（信阳市药检所中药实验室制水机，一级水）；其他试剂均为分析纯。22方法与结果221对照品溶液的制备取丹参酮A对照品106MG置25ML容量瓶中，精密称定，加甲醇定容。用1ML移液管移取配置好的对照品溶液，另置25ML容量瓶中，加甲醇定容，即得。配得的对照品溶液浓度为1696UGML222供试品溶液及阴性样品的制备取本药品5份，每份32G，精密称定。分别置同型号250ML具塞锥形瓶中，各加入25ML甲醇。精密称定。超声提取30MIN。放至室温，精密称定，用甲醇补足损失的量。过滤，取续滤液，即得。模拟处方的制备方法，制备不含丹参的阴性样品，按供试品制备方法制备成阴性样品溶液。表1取样量超声前超声后补足后1号实验318731163690116326911636422号实验3177011229921123287111229313号实验319339430759429459430214号实验321881043507104334810435205号实验31842881713881548881748223色谱条件色谱柱AGILENTC18柱（250MM46MM,5UM）；流动相以甲醇水（2080）为流动相；流速10MLMIN1；检测波长270NM；柱温25。理论板数按丹参酮A峰计算应不低于2000。分离度15；龙芪柔肝胶囊中其他成分对丹参酮A含量的测定无影响。1对照品2供试品3缺丹参对照品224线性关系的考察精密吸取配置好的丹参酮A对照品溶液1UL、3UL、5UL、7UL、9UL注入高效液相色谱仪，依法测定，以峰面积积分值为纵坐标（Y），以进样量（UG）为横坐标（X）,绘制标准曲线。结果表明丹参酮A在169615264UGML范围内呈良好的线性关系。回归方程为Y5759589X101938R2099969进样量进样量（G）保留时间峰面积面积1微升155112573000075463微升15537183679000082315微升15511383743000091797微升15504580916000097889微升1549678100300009191Y575958924336X10193821441R209996910000000000000000100000000002000000000030000000000400000000005000000000060000000000700000000008000000000090000000000000000002000004000006000008000010000012000014000016000018000进样量峰面积225精密度试验取中间上述25对照品注入高效液相，重复进样6次，每次进样5UL。记录丹参酮A峰面积，结果丹参酮ARSD169N5226重复性试验取同一批号供试品，按23项下方法制备5份供试液，分别进样，测定峰面积，计算丹参酮A的平均含量为0081G1，其RSD267（N5）。227稳定性试验取同一供试品溶液，分别在0、3、6、9H按上述色谱条件试验，记录丹参酮A峰面积积分值，结RSD3；表明供试品溶液在9H内基本稳定。228加样回收率试验精密称定已测定含量的样品（1696UGML），共取5份，每份32G，按22项下方法制备供试品溶液。分别精密加入丹参酮A对照品储备液1ML，进样。计算丹参酮A含量并计算回收率为10132RSD28N5。229样品的含量测定取3批龙芪柔肝胶囊样品，按上述供试品溶液的制备方法制备，并按上述色谱条件测定丹参酮A含量。结果0081G13讨论31检测波长的选择经过AGILENT1200三维图谱分析，发现在270NM,275NM处均有吸收峰，且吸收系数均较大，考虑到龙芪柔肝胶囊药味比较复杂，对丹参酮A的检测容易产生干扰，因此选择检测波长为270NM。32色谱条件的选择曾用甲醇水（2575）作为流动相1，但丹参酮A峰形有拖尾，用于定量分析不准确。经过试验，调整流动相为甲醇水（2080）。峰形明显改善。影响峰形的因素是否还与其他因素（如PH）有关，尚需进一步研究。33提取方法的选择331药品预处理取龙芪柔肝胶囊4份，每份32G，精密称定。分别置250ML具塞锥形瓶中。编号为回1、回2、超1、超2。各瓶精密加入甲醇50ML,称定重量。332供试品溶液的提取对回1进行30MIN回流提取，对回2进行45MIN回流提取。对超1进行超声30MIN超声提取，对超2进行45MIN超声提取1。333供试品的筛选将上述四瓶供试品放至室温，精密称定，补足损失的量，过滤。取续虑液，用HPLC进行检定。发现30MIN时即提取完全。且超声提取优于回流提取。4参考文献1国家药典委员会中华人民共和国药典一部S北京中国医药科技出版社,20102张德钦中药分析制剂技术北京中国中医药出版社3方泰惠吴清和中药药理学北京科学出版社4陈琼中药制剂技术北京中国农业大学出版社5王均宁薛建国方剂学北京科学出版社6凌一揆中药学上海上海科学技术出版社7唐丽琴，张善堂，陈象青，等HPLC法测定不同来源补骨脂中补骨脂素和异补骨脂素的含量M中国药师，2011，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