锥形角转子高效逆流色谱新方法及其在天然产物分离和识别中的应用

Despite some biosynthesis or combination chemistry-based methods has been used to compose a large number of chemical entities or compounds library in recent years, natural products are still the most consistently successful source of drug leads. However, the extracts from natural resources like plants, animals or macromycetes are very complex. They usually contain complex chemical components and possess versatile chemical structures. It is still a challenging task for isolation of natural products from natural resources. It has been known that counter-current chromatography is a unique liquid-liquid partition chromatography without support matrix, which eliminates several complications resulting from the solid support matrix, such as irreversible adsorption and denaturation of samples. Thus, counter-current chromatography is very suitable for natural products isolation. Unfortunately, so far the separation efficiency of counter-current chromatography is not very satisfactory. Recently, we developed a counter-current chromatography apparatus with three tapered separation columns, named conical counter-current chromatography, which demonstrated higher retention of stationary phase and better resolution due to that the tapered column support makes centrifugal field to distribute gradiently in radical and axial directions. It seems true that the distribution of centrifugal fields in the radical and axial directions will increase gradiently when the separation column is slanted. Therefore, in this work, we aim to fabricate a new conical angel rotor high-performance counter-current chromatography with appropriate angles of taper and inclination in order to retain more stationary phase and achieve higher resolution. In addition, we will isolate several target compounds from the crude extracts using the developed conical angel rotor counter-current chromatography. Furthermore, we will develop a new strategy for pattern recognition and discovery of known and unknown components from complex natural product mixtures by combining conical angel rotor high-performance counter-current chromatography fractionation with 13C NMR pattern recognition. In summary, in this work, we will establish and develop conical angel rotor high-performance counter-current chromatography and a series of application methods will be developed for natural products isolation. It is essential and very useful for current drug research and developments.

天然产物仍是新药研究的重要源泉。尽管许多天然来源的化合物可以通过化学或生物的方法方便地合成和制造，但如何纯化和识别具有生物活性的新型天然产物仍是一个重要的挑战。前期研究发现，绕制成锥形的逆流色谱分离柱可形成梯度径向和轴向的离心力场，有利于固定相的保留和分辨率的提高。本项目拟在此基础上，更进一步，构建一种高效的分离柱呈倾斜角排布的锥形角转子逆流色谱仪器和方法，在不影响逆流色谱混合效率的情况下增加其固定相保留率，提高其分辨率。同时，应用这种锥形角转子高效逆流色谱，建立和发展一系列定向分离天然产物目标组分的方法，构建一种逆流色谱分离—NMR模式识别发现和鉴定新型天然产物的策略，发现和定向分离制备出一批具有药效活性的新型天然药物成分。此举不仅可有效地改善和提高现有逆流色谱的分离效率，拓展其应用范围，而且将有助于丰富现有色谱的理论和应用，加速天然药物研发的进程，推动相关学科和产业的创新发展。

尽管各种新的高效的分离分析方法不断涌现，逆流色谱由于其独特的基于全液液相分配原理的无固相载体吸附等优点，迄今仍是天然产物研究中的一种重要的分离制方法。本项目在前期研究基础上，通过将现有J型逆流色谱的分离柱呈锥形绕制和倾斜角布置，并结合行星运动同步解绞原理，而设计构建了一种锥形角转子逆流色谱。经过参数考察和优化，基本表明这种方法在优化的锥角和倾斜角及绕线方式的情况下，可以改进固定相的保留和提高分离效率，对于一些样品，可以接近于柱色谱的组分分辨率。在此基础上，初步建立了几种定向分离天然产物的模式和联用方法；并利用代谢组学方法和技术，构建了一种逆流色谱分离—NMR模式识别发现和鉴定天然产物的方法，并以八角莲、丹参、厚朴、荜菝等特色中药材为原料，发现和定向分离制备出一批具有一定活性的抗癌天然产物成分。同时，本项目围绕研究计划中的仪器构建、分离模式和模式识别等三个内容还做了较多的拓展，开发了一系列的分离制备方法，并发展了一系列新型天然低共熔溶剂，这为下一步逆流色谱绿色溶剂的开拓提供了思路。因此，尽管还有相当多的工作仍在进行，一些工作还处于雏形，一些工作还待发表，但现有这些研究，已经明显地表明这种锥形角转逆流色谱仪器方法是一类有效的仪器方法设计，一方面不仅可有效地改善和提高现有的逆流色谱的分离效率，拓展其应用范围，而且将有助于丰富现有逆流色谱的理论和应用，加速天然药物研发的进程，推动相关学科和产业的创新发展。