【作者】 周蓓蓓；

【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】 苯丙素类酚酸性化合物药理活性突出,如丹酚酸等,越来越受到药学界的重视。苯丙素类酚酸结构均含有苯丙基(Ph-C-C-C-),分离特性相似,存在稳定性差,检测条件苛刻,分离耗时长等问题,尤其是成分复杂的多酚酸混合样品,往往需要梯度洗脱法测定,与酚酸性质相近的杂质(如黄酮苷类,同分异构体等)干扰现象也很严重。本文围绕苯丙素类酚酸色谱分离的关键技术难题,以典型化合物为对象,研究其快速配位色谱分离与分析方法。选择了多种配体,通过计算机模拟,化学计算和实验分析探讨了目标化合物与配体的配位机理,分析了配体的适用性,总结了苯丙素类酚酸的配位色谱分离规律,其研究结果如下:1.如果分离测定一对常规方法较难分离的苯丙素类酚酸,要根据它们结构的差异来选择相应的配位色谱法,现将可能出现的结构差异分为两大类:(1)存在支链双键差异,(2)存在羧基差异。这种差异是基团数量或位置差异。当一对酚酸分子既有双键差异又有羧基差异的时候,要根据分子结构确定哪种差异占主导地位,一般来说优先选用硝酸银配位色谱法。2.选择硝酸银,氯化钙,硼砂,尿素作为配体,采用ChemBioOffice 2008软件和依据分子力学(MM2)能量最小原则,对6种典型酚酸(丹参素,咖啡酸,菊苣酸,丹酚酸A,丹酚酸B,迷迭香酸)与4种配体配位前后体系的最小总能量进行计算,根据计算结果得到结论:对于金属配体,硝酸银的配位能力略强于氯化钙,对于非金属配体,硼砂和尿素的配位能力基本相同。几种配体配位能力强弱顺序为:Ag+>Ca2+>尿素≈硼砂。3.当一对酚酸结构差异符合(1)时,宜采用银离子双键配位进行样品的色谱分离,配体优选硝酸银。依此,建立了菊苣酸和单咖啡酰酒石酸,丹酚酸B和异丹酚酸B这两对酚酸的硝酸银配位色谱分离方法,硝酸银的浓度为6～9mmol/L,分离度分别为1.5和2.3。红外,质谱等分析表明,配位是多点(n≥3)联合作用,主配位点是双键,同时受到相邻苯环和羰基的强烈影响。4.当一对酚酸分子结构差异符合(2)时,宜采用钙离子羧基配位进行样品的色谱分离,配位剂优选氯化钙。依此,建立了丹酚酸A和B,菊苣酸和洋蓟酸这两对酚酸的配位色谱分离方法,氯化钙的浓度为5～8 mmol/L,分离度分别为1.9和1.7。主配位点是分子中的羧基,同时受到距离最近双键(直链双键或苯环大π键)电子云的强烈影响,配位是多点联合作用(n≥2)。苯环影响强于直链双键。5.当酚酸样品的杂质具有葡糖基结构,测定此样品时可选用硼砂抑制杂质,优化测定效果。硼砂溶解后生成硼酸,硼酸分子能够与葡糖基同侧三个或以上的羟基产生氢键平衡作用,形成空间结构契合型的氢键配位,这种配位不受pH值的影响,在酚酸测定的弱酸性条件下,这种配位大大强于四羟基硼络离子(硼砂溶解后的另一种产物)与酚酸邻二酚羟基的配位。更多还原

【Abstract】 Most of various phenolic acids are compounds which similar in structure and properties. The analysis and separation of them are fairly difficult, such as using gradient elution method. Phenylpropanoid phenolic acids are compounds of conspicuous pharmacological activities,which have“Ph-C-C-C-”in structure. It takes much time to determine and separate them and the analytical methods are always complicated. The determination is often disturbed by compounds such as isomers and flavonoid glycosides which are similar in characters to phenolic acids. This article was focused on the rapid determination and separation of several pink phenylpropanoid phenolic acids by complex chromatography and analysis of coordination mechanism.It was realized that the determination selectivity was improved by the coordination difference of coordination agent and target compounds. We chose different ligands for different groups and studied the coordination mechanism, coordination sites etc by ultraviolet spectrophotometry (UV), infrared spectroscopy (IR), mass spectrography (MS) and computer model. We found certain rules from the study for a wide range of uses.1. There is certain regularity in structure of phenylpropanoid phenolic acids. The most important groups for possible coordination were“C=C”and“-COOH”. If it is difficult to determine a couple of phenylpropanoid phenolic acids by routine method, we should choose suitable method according to the difference of their structure. There are two kinds of structure difference: (1) difference of“C=C”on side chain, (2) difference of“-COOH”. If a couple of phenolic acids have both“C=C”and“-COOH”difference, we should judge the more important difference from the specific structure. Generally speaking argentation complex chromatography is preferred.2. We chose four ligands: AgNO3, CaCl2, borax, urea and calculated the minimum binding energy before and after coordination according to the“minimum energy principle”(MM2)by the software of ChemBioOffice 2008, and we found that the minimum energy of coordination system was lower than before. We choosed two type of ligands: metal ion (silver nitrate, calcium chloride) and nonmetal (borax, urea). The mechanism of the two type ligands is different. For metal ligands, the ability of coordination of Ag+ is stronger than Ca2+; for nonmetal ligands, the ability of coordination of borax is approximately the same to urea. The proper order of their coordination ability is Ag+>Ca2+>urea≈borax.3. We chose silver nitrate as ligand for a couple of phenolic acids which have“C=C”groups difference. The determination methods of two couples of cichoric acid and caftaric acid, salvianolic acid B and isosalvianolic acid by adding silver nitrate to mobile phase were established. The concentration of silver nitrate was from 6 to 9mmol/l. The resolutions of them were 1.5 and 2.3 respectively which were qualified according to the determination requirements. The main coordination site was on“C=C”outside the benzene ring and the coordination was strongly influenced by electron cloud of benzene ring and carbonyl (on“-COOH”) nearby. The coordination was organized by n points force(n≥3).4. We chose anhydrous calcium chloride as ligand for a couple of phenolic acids which have“-COOH”difference in structure. The determination methods of salvianolic acid A and salvianolic acid B, cichoric acid and cynarin by adding calcium chloride to mobile phase were established. The concentration of calcium chloride was from 5 to 8mmol/l. The resolution were 1.9 and 1.7 respectively which were consistent with the standard of determination. The main coordination sites was on“-COOH”and the coordination was strongly influenced by electron cloud of“C=C”nearby (bigπbond of benzene ring was also involved). The effect of benzene ring was stronger than straight chain“C=C”.5. If the impurities of phenolic acids sample have glucosyls, we should choose borax as the ligand. Borax can act as the inhibitor of impurities which have glucosyls. At weak acidic situation, borax preferentially coordinated with compounds which have glucosyls. It was because that glucosyls had many hydroxyl groups which could form certain hydrogen bond balance with boric acid molecule when there were more than three hydroxyl groups on the same side. The hydrogen bond balance agreed with spatial structure and was not influenced by pH.更多还原