【作者】 金日生；

【导师】 潘见；

【作者基本信息】 合肥工业大学 ， 农产品加工及贮藏工程， 2009， 博士

【摘要】 天然产物中的生物碱类化合物种类繁多,结构复杂,常具有不同的药理作用,实用中多需分离纯化和分析检测。对极性差异小,常规反相色谱分离困难的生物碱进行快速、灵敏、可靠的定性、定量分析及分离一直是受人瞩目的研究课题。本文紧紧围绕生物碱类化合物色谱分离过程的关键技术难题,以三组同植物源生物碱为研究对象,进行配位色谱高效分析分离新技术研究。作者运用MM2分子力学方法计算生物碱类化合物与配位剂的相互作用,选择配位剂,优化配位色谱分离条件,构建色谱分离模型;进行配位色谱试验,实现目标化合物的分析检测。主要研究结果如下:1.提出生物碱类化合物配位色谱分离的配位剂选择策略,按生物碱的碱性、局部结构（位点）和整体（表观包络/形貌）结构差异的顺序选用配位剂。分离碱性差异的生物碱选择pH调节剂型配位剂;分离局部结构（位点）差异和整体（表观包络/形貌）结构差异的生物碱选择金属离子或超分子包合配位剂。2.建立了生物碱类化合物配位色谱分离配位剂的分子力学筛选方法。MM2分子力学方法计算不同生物碱配位前后的能量,基于配位后生物碱之间的能量差异大小选择配位剂,配位后造成能量差异较大的为优选配位剂。运用该方法计算选定了辣椒素和麻黄类生物碱的配位色谱分离的配位剂为Ag⁺和β-环糊精。3.调节流动相pH和有机溶剂比例实现了碱性差异石杉类生物碱的高效分离。考察了有机溶剂比例、pH值对分离的影响。生物碱的保留时间随有机溶剂比例的增大、pH值的减小而降低,通过改变流动相的有机溶剂比例、pH值可以实现碱性差异生物碱的高效分离。在石杉生物碱的色谱分离流动相中引入氨基酸,建立了碱性差异生物碱绿色配位色谱分离方法。4.建立了具有局部结构差异的辣椒素的高效液相Ag⁺配位色谱分析方法。0.03mol/L硝酸银添加到配位色谱流动相中实现了辣椒素的高效分离。电喷雾质谱实验研究表明气相条件下存在辣椒素和Ag⁺的加合物,辣椒碱-Ag⁺的稳定性强于降二氢辣椒碱-Ag⁺和二氢辣椒碱-Ag⁺。通过计算化合物的logP值,表明辣椒碱-Ag⁺配合物的疏水性降低,和降二氢辣椒碱之间的差异增大,提高分离度。5.建立了具有整体结构差异的麻黄碱和伪麻黄碱的高效液相环糊精配位色谱分析方法。0.01 mol/Lβ-环糊精添加到流动相中实现了麻黄碱和伪麻黄碱的高效分离度。随着配位剂β-环糊精浓度的增加,麻黄碱和伪麻黄碱的保留时间降低。通过考察β-环糊精浓度对容量因子的影响,表明配位色谱液相色谱条件下环糊精与麻黄碱及伪麻黄碱之间形成了1:1配合物,基于生成的包合物的稳定性差异实现它们的高效分离。麻黄碱和伪麻黄碱与β-环糊精形成的包合物的包合常数分别为7.35、41.30（mol/L）^-1。6.生物碱在不同配位剂的配位色谱分离中具有相同的保留机制。生物碱能和配位剂形成1:1配合物时,其容量因子（?）（其中k₁、k₂为生物碱和生物碱配合物的容量因子,Kb为形成配合物的稳定常数,L为配位剂）。更多还原

【Abstract】 Active components in natural products possess various pharmacologic effects and receive considerable attention in the literature. Isolation, purification and analysis were essential in their routine application. Thousand species of alkaloids was discovered up to now, which are structure related compounds and great efforts are needed to conquer the complexation of separation. This assay was focus on the bottleneck of R&D procedure of alkaloids which have little differences between polarities. Separation method of three diverse model components was achieved by coordination chromatography technology.Interaction of target compound and coordination agents was studied using MM2 molecular mechanism method. Coordination chromatography experimental research was carried out accordingly and mathematical model of coordination chromatography was established. Different alkaloids were tested to investigate cooperative effectiveness of coordination agents to chromatographic separation. Separation mechanism of coordination chromatography was resolved preliminarily. Main research finding was following:1. The selection strategy of coordination agent to separate alkaloids which have little differences between polarities was proposed, according the order of differences between basicity, partial structure and integral structure .The pH regulator should be selected as coordination agent to separate the alkaloids which have differences between basicity; metal ion or supramolecular coordination agent such asβ-cyclodextrin （β-CD） should be selected as coordination agent to separate the alkaloids which have differences between partial structure and integral structure.2. The screening method of coordination agent to separate alkaloids was established using MM2 molecular mechanism method. The energy differences between alkaloids before and after coordination were calculated, the coordination agent which could make more difference was preferred.3. A selective HPLC method for determination of Huperzine A and analogues in Huperzia serrata Extract was developed by adjusting the pH value of mobile phase. The effects of pH value and concentration of organic solvent were investigated, the retention of alkaloids decreased with the concentration of organic solvent increasing or the pH value decreasing. The amino acids were introduced in the mobile phase, a green HPLC method for determination of Huperzine A and analogues was accomplished with L-lysine as a mobile phase modifier.4. Isolation method of capsaicinoids was accomplished by coordination chromatography using Ag⁺ as coordination agents. Ag⁺ adjusted chromatographic resolution of capsaicin and nordihydrocapsaicin. Capsaicinoids could form adducts with Ag⁺ at electron spray ionization mass spectrometry. The effect of Ag⁺ concentration on the retention and selectivity of ephedrine and pseudoephedrine was investigated. Graphs of 1/k vs. [Ag+] gave good linear relationships, indicating the inclusion ratio of 1:1 between Ag⁺ and capsaicin.5. Isolation method of ephedrine alkaloids was accomplished by coordination chromatography usingβ-CD as coordination agents.β-CD adjusted chromatographic resolution of ephedrine and pseudoephedrine. The effect ofβ-CD concentration on the retention and selectivity of ephedrine and pseudoephedrine was investigated. Graphs of 1/k vs. [β-CD] gave good linear relationships, indicating the inclusion ratio of 1:1 betweenβ-CD and ephedrine / pseudoephedrine.6. The alkaloids separated by coordination chromatography with different coordination agent have the same retention mechanism. When the alkaloid formed 1:1 coordination with coordination agent, the capacity factor （k） of alkaloid can be described with the following equation:, where k₁ and k₂ are the capacity factors of the alkaloid and the 1:1 coordination, respectively, L is the coordination agent.更多还原