【作者】 刘高；

【导师】 饶力群；

【作者基本信息】 湖南农业大学 ， 生化与分子生物学， 2008， 硕士

【摘要】 本研究以叶下珠为研究对象,建立了叶下珠中叶下珠素的高效液相色谱法（HPLC）检测方法,同时采用HPLC测定不同产地和不同部位的叶下珠药材中叶下珠素含量;对叶下珠中叶下珠素的提取与分离纯化工艺进行了较深入而系统的研究,确定了工艺流程和分离纯化条件,同时对纯化的叶下珠素单体进行了质谱鉴定。主要研究内容如下:HPLC定量检测叶下珠素的含量。确定液相色谱条件为:色谱柱C₁₈（250mm×4.6mm,5um）,检测波长230nm,柱温30℃,进样量20μL,流速0.8mL/min,以甲醇—水（80:20）为流动相等梯度洗脱。在此液相条件下,叶下珠样品液中五种组分得到较好地分离。叶下珠素在2.72μg/mL—136μg/mL浓度范围内线性关系良好。HPLC测定不同产地和（广西,南宁）不同部位叶下珠药材中叶下珠素含量。结果表明,海南产叶下珠中叶下珠素含量最高为0.664%,广西次之（0.565%）,广东,湛江为（0.178%）,其他产地叶下珠素含量均较低。不同部位以叶中含量最高（1.04%）,果实（0.03%）、茎（0.02%）、枝（0.018%）、根（0.01%）中含量都很低。叶下珠中叶下珠素的提取工艺研究。乙醇超声法提取,进行单因素试验和正交试验,确定最佳提取工艺参数。结果表明,影响叶下珠素提取效率的各因素主次顺序是:乙醇浓度＞液料比＞提取时间,提取温度的影响高度显著。最佳提取工艺参数为:乙醇浓度95%,液料比25,提取时间20min,重复提取两次,叶下珠素含量为0.687%。叶下珠素的纯化工艺研究。比较HPD100、HPD100A、HPD700等七种大孔树脂对叶下珠素的吸附分离特性,确定选择HPD100大孔树脂。较优纯化条件为:上样流速为1BV/h,选择80%乙醇为洗脱溶剂,洗脱流速为2BV/h,洗脱液用量约为9BV,在该条件下叶下珠素纯度从9.47%提高到55.2%通过硅胶柱层析纯化叶下珠素粗产品得到纯度为83%,选择石油醚对叶下珠素粗品进行重结晶得到了纯度为96.79%。对纯化的叶下珠素单体进行了质谱鉴定,结果表明纯化所得晶体与叶下珠素标准品质谱图基本一致。更多还原

【Abstract】 Phyllanthus urinaria L.as the raw material,established the HPLC detects the content of phyllanthin by quantitative an alysis,the content of phyllanthin in different areas and parts of phyllanthus urinaria L.were measured;Deep and systematical researches had done to the extraction、isolation and purification of phyllanthin from phyllanthus nurinaria L.,isolation and purification factors were studied and established;The purification of phyllanthin was identified by mass spectra.The main research content is as folows:The HPLC detects the content of phyllanthin by quantitative analysis.The conditi ons of deter mining liquid phase chromatogram are as folows:chromatogr am column C₁₈（250mmx4.6mm,）,the wavelength of detector at 230nm,tempera ture at 30℃,sample size20μL,rate at 0.8mL/min,met hanol:water（80:20v/v）as mobile Phase.In this case,at least 5 components alcohol extactive can be separated well from impurities.Good linear relatio nships between the peak area values and concentrations have beenfound.The relationship of the phyllanthin is good in the range of 2.72μg/mL-136μg/mL.The content of phyllanthin in different areas and parts of phyllanthus urinaria L.were measured by HPLC.The results showed that the content of phyllanthin in phyllanthus urinaria L.from Hainan was 0.664%,Guangxi and Guangdong zhanjiang were 0.565%and 0.178%,the content of phyllanthin in leaf,fruit, steam,root and branch were 1.04%,0.03%,0.02%,0.018%,0.01%.The extraction technics of phyllanthin was extracted from Phyllanthus urinaria L.by ethanol ultrasonic extraction,through oneway and orthogonal experiment the optimum parameters of extracting phyllanthin had been determined. It was showed that the main factors influencing extraction efficiency were as follows:the ethanol concentration＞the ethanol amount＞extraction times.The optim um parameters was:the concentration of ethanol is 95%,the multiple of ethanol to material is 25,extraction time is 20min,by two extractions,the content of phyllanthin was 0.687%.The purifying technology of phyllanthin.The study compared adsorption properties of seven macroporous resins,includingHPD100,HPD100A,HPD700, D101、D201、NKA-Ⅱ、NKA-9、HPD 100 resin was determined as the best sorbent.The optimum purification condition o btained was:the driedre sina sre ference,the flow rate on absorption is 1BV/h,9BV80%ethanol,the flow rate on desorption is 2.0BV/h.Under these conditions,the purity of phyllanthin has greatly improved from 9.47%to 55.2%,Through silica gel chromatographic column to purify phyllanthin from overall phyllanthin,the purify of phyllanthin obtained was 83%.Phyllanthin was purifid from overall phyllanthin to obtain pure phyllanthin by crystal and recrystal means.its purify was up to 96.79%.Purification of phyllanthin under-the-MS identification,the results showed phyllanthin was purified from phyllanthus urinaria L.and standards of quality spectrum basically the same.更多还原