银杏叶中银杏内酯的分离纯化及标准样品研制

发布时间：2017-12-28 20:23

本文关键词：银杏叶中银杏内酯的分离纯化及标准样品研制　出处：《山东农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：银杏(Ginkgo biloba L.)是一种裸子植物落叶乔木,我国自古以来就有银杏叶入药和食疗的记载,20世纪70年代初在国外特别是欧洲利用银杏叶提取物(extract of Ginkgo biloba,EGB)来治疗心脑血管疾病包括脑血管动脉粥样硬化和中枢外周血流紊乱的研究出现,其药用价值受到了更为广泛的关注。银杏叶提取物是经现代提取工艺从银杏叶中提取的活性物质,其主要活性化学成分主要包括两大类:黄酮类及萜类。银杏内酯A、B、C等是二萜类内酯,白果内酯属于半萜类内酯,是血小板活化因子(platelet activating factor,PAF)拮抗剂。临床上使用银杏内酯治疗心血管疾病、急性胰腺炎和血栓等。本研究主要进行了银杏内酯的分析方法、提取工艺的优化,并进行高纯度银杏内酯制备分离,得到的银杏内酯样品进行定值分析,证明结果符合标准样品的要求,可以使用。主要研究成果如下:1.银杏内酯分析方法的优化:采用Agilent 1260串联385-ELSD检测器及YMC-Pack ODS-A(5μm,4.6×250mm)的反相色谱柱进行分析,依据分离度最大(实际测得分离度R7.2)、检测时间适宜、流动相安全等要求,优选异丙醇:水(10:90,v/v)作为流动相,流速为1ml/min,气体流速为1.60L/min(N2),漂移管稳定温度为90℃,可以更加安全准确地定量测定银杏内酯。2.优化银杏内酯提取方法:在单因素试验的基础上,采用四因素三水平的响应曲面分析法,建立乙醇提取银杏叶中银杏内酯得率的二次多项数学模型,并以银杏内酯提取率为响应值作响应曲面和等高线,考察提取次数、提取时间(h)、乙醇的浓度(%)和固液比(g/ml)对银杏内酯提取率的影响。结果表明,银杏内酯提取的优化工艺条件为:提取次数为3次,提取时间2h,乙醇浓度44%,固液比0.04g/ml,在此工艺条件下,银杏内酯的提取率为0.46%。同时对比亚临界水提取效果,为了提高银杏内酯的提取率及提取效率,进行单因素实验分析四种因素:提取温度,提取时间,提取次数和料液比对银杏叶中银杏内酯提取率的影响并确定因素水平。运用响应面实验方法优化亚临界水提取银杏叶中银杏内酯的工艺,并对各影响因素的重要性进行排序。研究表明当提取温度180℃,料液比(g/ml)为1∶25,提取时间30min,提取次数为3时,银杏内酯的提取率为0.47%。亚临界水萃取具有绿色环保、无有机溶剂残留、提取率高等特点。3.银杏内酯的前处理。经过提取得到银杏内酯粗提物,用石油醚进行脱脂至石油醚相接近无色,用乙酸乙酯萃取。乙酸乙酯提取液加入弱碱NaHCO3溶液洗涤3次脱除黄酮类物质。硅胶脱除极性较大的小分子杂质比活性炭,硅藻土,酸性氧化铝吸附效果好。利用重结晶法对银杏内酯B进行纯化,在实验中用乙醇和水对银杏内酯进行重复结晶可以得到银杏内酯B的纯度为95%。4.高速逆流色谱法纯化银杏内酯,选择溶剂系统为正己烷-乙酸乙酯-甲醇-水(4/5/3/5,v/v/v/v),正向旋转,转速为800r/min、流速为5ml/min、操作温度25℃得到白果内酯、银杏内酯A、B、C制备纯度为99%以上。5.对银杏内酯进行结构鉴定。通过薄层色谱、质谱仪、核磁共振图谱等对银杏内酯结构进行鉴定。6.对样品进行定值分析。本品按照GB/T15000.3-2008标准要求,采用多个实验室协作试验定值,参加实验室的数目为8个。按照标准样品定值程序,用高效液相色谱仪测定,采用峰面积归一化法进行纯度定值,经过统计计算得到样品的标准值及不确定度。
[Abstract]:Ginkgo biloba (Ginkgo biloba L.) is a species of gymnosperms, deciduous trees, since ancient times China's ginkgo leaf is used as medicine and diet records, at the beginning of 1970s in foreign countries especially in Europe using the extract of Ginkgo biloba (extract of Ginkgo biloba, EGB) to the treatment of cardiovascular and cerebrovascular diseases including cerebral vascular atherosclerosis and central peripheral blood flow disorder there, its medicinal value attracted more attentions. Ginkgo biloba extract (Ginkgo biloba extract) is an active substance extracted from the leaves of Ginkgo biloba by modern extraction technology. Its main active chemical components include two major categories: flavonoids and terpenoids. Ginkgolide A, B, C and so on are two terpene lactone, and the ginkgo lactone belongs to the terpene lactone, and it is a platelet activating factor (platelet activating factor, PAF) antagonist. Ginkgolide is used clinically for the treatment of cardiovascular disease, acute pancreatitis and thrombus. This study mainly carried out ginkgolide analysis methods, optimization of extraction technology, and made high-purity Ginkgolides separation and ginkgolide samples obtained by fixed value analysis. The results showed that the results accorded with the requirements of standard samples, and they could be used. The main research results are as follows: 1. analysis methods for optimization of Ginkgolides: using Agilent 1260 385-ELSD detector and YMC-Pack ODS-A series (5 m, 4.6 x 250mm) reversed-phase HPLC column was analyzed, based on the separation of the largest (measured resolution R7.2), mobile phase, detection time for security requirements, selection of isopropyl alcohol water: (10:90, v/v) as mobile phase, the flow rate was 1ml/min, the gas flow rate is 1.60L/min (N2), stable drift tube temperature is 90 DEG C, can be more safe and accurate quantitative determination of ginkgolides. 2. optimization of Ginkgolides extraction method: on the basis of single factor experiment, the response surface of four factors and three levels of analysis, two multinomial mathematical model of ethanol extraction of Ginkgolides in Ginkgo leaf yield, and the extraction rate of Ginkgolides as response surface and contour as the response value, the effects of extraction times and extraction time (H), ethanol concentration (%) and solid-liquid ratio (g/ml) effect on the extraction rate of ginkgolides. The results showed that the optimum extraction conditions of Ginkgolides were: extraction times 3 times, extraction time 2h, ethanol concentration 44%, solid-liquid ratio 0.04g/ml, under this technological condition, the extraction rate of Ginkgolides was 0.46%. At the same time, the effect of subcritical water extraction was compared. In order to improve the extraction rate and extraction efficiency of Ginkgolides, a single factor experiment was conducted to analyze four factors: extraction temperature, extraction time, extraction times and ratio of material to liquid, and determine the level of Ginkgolides. The process of extracting ginkgolide from Ginkgo biloba leaves by subcritical water was optimized by the response surface method, and the importance of the factors was sorted. The results showed that the extraction rate of ginkgolide was 0.47% when the extraction temperature was 180, the ratio of material to liquid (g/ml) was 1: 25, the extraction time was 30min, and the extraction times were 3. Subcritical water extraction has the characteristics of green environmental protection, no residual organic solvent and high extraction rate. 3. pretreatment of ginkgolide. The crude extract of ginkgolide was extracted, and oil ether was used for degreasing to petroleum ether, which was colorless and extracted with ethyl acetate. Ethyl acetate extract was washed with weak alkali NaHCO3 solution for 3 times to remove flavonoids. The adsorption of silica gel with high polarity is better than that of activated carbon, diatomite and acid alumina. Ginkgolide B was purified by recrystallization. In the experiment, ethanol and water were used to recrystallize Ginkgolides, and the purity of ginkgolide B was 95%. Purification of ginkgolide 4. high-speed countercurrent chromatography, solvent system selection for hexane - ethyl acetate - methanol - water (4/5/3/5, v/v/v/v), the positive rotation speed is 800r/min, the flow rate was 5ml/min, the operating temperature of 25 DEG C by bilobalide, ginkgolide A, B, C preparation purity was more than 99%. 5. the structure of ginkgolide was identified. The structure of ginkgolide was identified by TLC, MS, NMR and so on. 6. to analyze the value of the sample. In accordance with the GB/T15000.3-2008 standard, this product adopts a number of laboratory cooperation tests, and the number of participants in the laboratory is 8. According to the standard sample calibration procedure, the HPLC method was used to determine the purity, and the peak area normalization method was used to determine the purity. After that, the standard value and uncertainty of the samples were obtained.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ464

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