【作者】 钟世安；

【导师】 周春山；

【作者基本信息】 中南大学 ， 应用化学， 2002， 博士

【摘要】 论文以儿茶素中各成分的反相液相色谱(RPLC)行为差异为基础，系统地研究了分析型、半制备型HPLC的计量置换保留模型(SDM-R)及其各参数的表征，在此基础上，对半制备型LC进行放大，探寻色谱分离条件的放大规律，得到大型制备型液相色谱分离天然酯型儿茶素的色谱条件，并成功地从中低档茶叶提取物-多酚类混合物中大批量同时分离提取了一类具有防癌、抗癌、抗衰老、降低血脂、提高免疫力和保健等一系列重要药理活性的极有价值的高纯天然药物中间体-天然酯型儿茶素单体(EGCG，GCG，ECG)。所提取的上述药物中间体通过HPLC、UV、FT-IR、HNMR及MS等手段进行定性、定量分析并确证了其结构。主要内容及结果摘要如下： (1)计量置换模型的建立及各参数表征 根据液固吸附体系中溶质—溶剂、溶质—吸附剂、溶剂—吸附剂、溶剂—吸附剂络合物的再溶剂化、溶剂化溶质—吸附剂络合物解吸共五个热力学平衡，推导出儿茶素的计量置换吸附模型(SDM-A)：10g P_c=β_a-q/Z log[PDm]，其中P_a为溶质在液固两相中的分配比，PDm为溶质在液相中的摩尔浓度。计量置换吸附模型参数(SDM-A)中q/Z可作为液固吸附体系的吸附效率的评价；儿茶素的计量置换保留模型(SDM-R)：1gK’=1gI-Z 1gα_D；其中Z为溶剂化溶质与溶剂化固定相相互作用时从溶质与固定相表面上释放出的溶剂分子的数目，1gI为1mol溶质对固定相的亲和势。对于溶质为非同系物的色谱体系，随着同系物置换剂溶剂分子的增大，溶质的上述二元计量置换参数依次减小，并呈现出线性变化；由SDM-R二参数Z、1gI作图，所得到的斜率J在数值上符合碳数规律，因而J可用于RPLC中表征溶剂强度。关于溶质为非同系物的SDM-R以及SDM-A参数q／Z的表征目前尚未见文献报道。 (2)半制备及制备型色谱液相色谱分离纯化酯型儿茶素 采用自行设计的半制备及制备色谱分离体系，实现了多酚类混合物中各主要酯型儿茶素的同步分离；在分离过程中，筛选出一种绿色添加剂溶剂B-1，改善了分离度，并防止酯型儿茶素(EGCG，GCG，ECG)氧化和缩聚合，制备出高纯度的酯型儿茶素单体。制备型色谱液相色谱分离纯化酯型儿茶素的最佳色谱条件如下： 制备型100×600mm色谱柱分离天然酯型儿茶素的最佳色谱条件为： ODS固定相(粒径10-30μm)；流动相：溶剂A/溶剂B-1(羧酸类)／水中南大学博士学位论文摘要== 14.5:0.5:85(v/v);体积流速为72.smL/min;进样量:4200mg;进样体积(v、/mL)二12mL;检测波长:280nln. 在此条件下收集馏分，各单体的回收率分别如下:EGcG，93%;GcG，95%; 而EcG峰是纯组份的峰，回收率98%。(3)大型制备型液相色谱的色谱条件的放大规律 制备型液相色谱中，1gK’与lga。具有良好的线性关系。对于色谱柱大小相同而色谱固定相不同，1 91及Z亦不同，且同类固定相粒径越粗，191及Z越小;对于色谱柱大小不相同色谱固定相相同，191及Z相差不大。对于色谱固定相相同的色谱体系，由半制备型液相色谱放大到大型制备型液相色谱，色谱条件的变化规律如下:a，溶剂配比基本不变化;b，最佳体积流速与色谱柱横截面半径的平方成正比，但由于涡流扩散，使得最佳体积流速在放大过程中实际值较理论值偏小;。，样品的进样量及其进样体积随色谱柱固定相有效体积的增大而增大，并且两色谱柱的进样量之比与两色谱柱容积之比一致。关于制备型液相色谱的计量置换保留模型及其参数表征，大型制备型液相色谱的色谱条件放大规律研究等尚未见文献报道。(4)天然酷型儿茶素的工业化制备研究 多酚类混合物经过纯化粗分工艺，色谱分离工艺，树脂吸附洗脱工含，等流程，可得到高纯度的天然酷型儿茶素EGCG、GCG、ECG单体。所提取的大然醋型儿茶素单体均通过HPLc定性、定量分析及uv、IR、HNMR、Ms等手段确证了其结构。整个分离制备过程不使用有毒害作用的溶剂，从源头上消除了有毒残留物对产品的污染。所得到的醋型儿茶素单体纯度高，均达99.2%以上，远高于国内外同类产品纯度要求与质量要求(51 glna公司同类产品最高纯度为95%)。(5)溶剂的回收工艺 在制备色谱分离纯化天然醋型儿茶素时，我们采用了减压膜蒸馏技术和自行研制的膜蒸馏装置处理废流动相，从中回收溶剂A，所得最佳工艺条件如下: PVDF微孔膜，冷侧压力0.013MPa，料液温度65℃，料液流速55L·h一，，料液中溶剂A浓度25%(V/v)。在此条件下膜具有良好的分离性能，此时，膜通量为35.08L·m-，·h一‘，馏出液溶剂A浓度为34.02%(v/v)，儿茶素截留率99.67%。该方法膜通量大、成本低、分离效果好。试验表明，该法对热敏性天然药物的浓缩分离，溶剂的回收等具有实用价值。(6)天然酷型儿茶素单体的体外抗癌药敏性试验 初步研究了天然酷型儿茶素(EGCG，GCG，ECG)单体抗消化道癌症一胃癌的体外药敏性试验，结果如下:MTT法测定研究表明，天然酷型儿茶索EGCG、GCG、中南大学博士学位论文摘要ECG单体对人胃癌细胞均存在浓度效应。比较而言，天然醋型儿茶素单体的抗癌活性顺序为EGcG、GCG>EcG;除EcG外，其他化合物的最大剂量组的抑瘤活性均在80%以上。 实?更多还原

【Abstract】 Based on the difference of Reversed-phase Liquid Chromatography behavior for catechins, Stiochiometic Displacement Model for Retention (SDM-R) of analytic LC and semi-preparative LC were studied, and the model parameters were studied also in this paper. On this base, semi-preparative LC was magnified. The magnified rule of chromatography conditions from semi-preparative LC to preparative LC was studied and three kinds of natural ester catechins which have important pharmacological function such as preventing and anti-cancer, resisting senescence, decreasing blood fat, increasing immunity ability and caring health were obtained. Main content and results are as flows:(1) Establishment of SDM and parameters’ tokenSDM-A and SDM-R of catechins were deduced according to five thermodynamics equation which are solute-solvent, solute-sorbent, solvent-sorbent, re-solventention of solvent-sorbent complex, desorbtion of solventen solute-sorbent complex in the liquid-solid system. SDM-A was: Ig PC = pa- q/Z lg[PDm], PC was distribution ratio of solute; [PDm] was Moore concentration of solute in liquid phase.SDM-R was: lg.K"’ = lgI -Z lgaD. Z was solvent molecular number that wasreleased from the surface of solute and stationary phase; Igl was affinity of 1 mol solute to stationary phase. The duality Stiochiometic displacement parameters of solute are decreased and presented linear change along with the increase of solvent molecule of homologue displacement. The numerical vaule of slope J was corresponding with carbon number role and can token solvent intensity. The study of non-homologue solute was not reported in literature.(2) Separation and purifition of ester catechins with preparative LCThe main ester catechins could be separated together. During the separation process, green additive solvent B-lwas added to improve resolution, prevent oxidation and polycondersation. For 100X600mm chromatography clumn, the composition and velocity of flow was optimized. The chromatography conditions were as follows: ODS (grain size 10-30μM) as stationary phase; solvent A/ solvent B-l/ water is 14.5:0.5:85(v/v), the rate of flow was 72.5mL/min, sample capacity wasIV4200mg. Under the chromatography stations, the recycling ratio of EGCG, GCG, and EGG is 93%, 95%, and 98% respectively.(3) the magnifying rule of chromatography conditionsIgl had good linear relationship with Igao in the preparative chromatography system. For the chromatography columns that had the same size but different stationary phase, Igl and Z were different. And for the columns with different sizes but the same stationary phase, Igl and Z were almost equal. When magnified to preparative chromatography from semi-preparative chromatography, the rules of change for chromatography conditions were as follows: a) the composition of solvent didn’t change for the different type; b) the optimum liquid speed was corresponding with the square of column radius, but the real value was smaller than the theoretical value because of the eddy diffusion; c) both volume and capacity of samples were homologous with valid volume of stationary phase.SDM-R of preparative LC and parameters token, the magnify rule of chromatography conditions was not reported in literature.(4) Industrialization preparation study of natural ester catechinsHigh pure natural ester catichins monomers (EGCG, GCG, ECG) were obtained through the series of techniques flow such as purification, chromatography separation, adsorption and desorbtion of polyphenols mixture. All the monomers were qualified and quantified with UV, FT-IR, HNMR and MS means. During the whole separation process, poisonous solvent was not used and poisonous hangover was avoided from headstream. The purity of monomers was higher than 99.2%, which was beyond the requirement of purity and quality for the same kind of product inside and outside.(5) Recycling technique of solventIn every technique, solvent was recycled and there was no environmental pollution problem. Waste mobile phase, which was更多还原