摘要
甘草是我国一种传统的中草药，其主要成份甘草酸（glycyrrhizin 或glycyrrhizic acid, GL）具有抗炎症，抗病毒，抗肿瘤，抗过敏等作用，而且它是一种甜味剂，甜度约是蔗糖的170倍。单葡萄糖醛酸甘草次酸（glycyrrhetic acid 3-O-mono-β-D-glucuronide, GAMG）作为GL的衍生物，两者具有相似的生物活性，其在抗炎和抗过敏活性上等同或超过GL，而且具有显著的抗癌作用。GAMG 是一种新型的天然甜味剂，甜度约是GL的5倍。因此，GAMG具有重要的研究价值。近年来，生物转化GL而直接制备GAMG成为研究热点。本论文围绕GAMG的酶法制备工艺及GAMG的分离纯化工艺展开系统的研究。主要工作总结如下：
1）建立了一种能同时检测生物转化体系中GL和GAMG的常规等度洗脱反相高效液相谱的定量分析方法。采用Apollo C18分析柱，流动相是V(甲醇)：V(水)：V(冰醋酸)＝75：25：5，流速1.0 mL/min，紫外检测波长254 nm，柱温为室温。在进样量0.2～20 μg内成良好的线性关系，GL的加样回收率为97.47%~99.32%，RSD为0.88%~3.78%；GAMG加样回收率为92.68%~98.44%，RSD为1.07%~3.95%。该法准确、简捷、耗材低廉。
2）GAMG制备工艺的研究。方法一：利用固态的酶颗粒对GL进行生物转化，经有机溶剂萃取后蒸干溶剂，得粗品GAMG；方法二：使用游离状态的酶及膜固定化反应装置对GL进行生物转化而制得GAMG的粗品溶液，经喷雾干燥后得其粗品。 3）系统地研究了树脂对GL及GAMG的吸附分离，主要包括以下内容：(1) 树脂的初步筛选。通过研究九种树脂对GL及GAMG的静态吸附，筛选出HP-20，NKA，X-5这三种非极性大孔吸附树脂及D380弱碱性阴离子交换树脂。 电视棒原理(2) 溶液的pH值对树脂吸附的影响。溶液的pH值会影响树脂的吸附容量，确定在进行吸附时溶液的pH值为5。
智能营销系统开发(3) 等温吸附行为的研究。将HP-20，NKA，X-5，D380这四种树脂对GL及GAMG 的吸附等温线用Freundlich方程进行拟合，其中X-5树脂对GAMG吸附拟合得到的方程的相关性最好；D380树脂对GL及GAMG均有较大的吸附容量；D380
中央排水系统
树脂对GL及GAMG吸附时的作用力最强，而NKA树脂对它们的吸附作用力最弱。比较HP-20，NKA，X-5，D380这四种树脂对GL溶液及GAMG溶液中的GL的吸附等温线，发现在吸附时溶液中GAMG的存在会与GL产生一定的竞争性吸附作用。
触摸屏调度台
(4) 静态洗脱行为的研究。用不同浓度的乙醇-水溶液作为洗脱液对上述吸附过溶液的四种树脂进行静态的洗脱，它们对吸附在HP-20，NKA，X-5树脂上的GL 及GAMG没有一定的选择性，而且不能将D380树脂上吸附的物质洗脱下来。使用不同浓度的NaOH、氨水溶液作为洗脱液，或者向其中加入不同浓度的NaCl 溶液或乙醇溶液作为D380树脂的洗脱液，实验表明其洗脱效果不理想，且没有一定的
选择性。综合考虑，选择HP-20，NKA，X-5这三种非极性大孔吸附树脂进行吸附动力学实验的研究。
(5) 吸附动力学行为的研究。NKA树脂对GL的吸附速度较NKA，X-5树脂要慢，在未达到饱和吸附前该树脂的吸附量相对较少，而这三种树脂对GAMG的吸附动力学曲线无明显差别，同时考虑到经济因素及NKA树脂的吸附交换系数明显大于其它两种树脂，选择NKA树脂进行动态实验。
(6) 动态吸附及洗脱行为的研究。对NKA树脂装填的交换柱进行动态吸附实验，绘制出贯穿曲线；用不同浓度的乙醇-水溶液对吸附过溶液的NKA树脂装填的交换柱进行动态的梯度洗脱，在乙醇-水溶液的浓度大于70%时，可以收集得到纯度为85.02%的GAMG，收率是35.36%。
PELOPHYLAX NIGROMACULATUS
4）采用加压型制备谱对树脂分离纯化得到的GAMG进行进一步的纯化。质谱及核磁共振谱确立该物质为GAMG。
关键词：甘草酸单葡萄糖醛酸甘草次酸大孔吸附树脂吸附解吸
ABSTRACT
Glycyrrhizic acid (or glycyrrhizin, GL) is a major ingredient of licorice (gancao
in Chinese) which is one of the most widely used medicinal herbs in China. Besides being a popular
food additive because of its sweetness that is about 170 folds of sucrose, GL is also known for its physiological activities, such as anti-inflammation, antitumor, anti-allergy and antivirus effects. Glycyrrhetic acid 3-O-mono-β-D-glucuronide (GAMG) is an important derivate of GL. It displays the similar (or stronger) anti-allergic and anti-inflammation effects with (or than) GL, and
it has the evident antitumor effect. The sweetness of GAMG is about 5 folds of GL. From above, it is anticipated that GAMG could be used as a new food additive and a therapeutic agent. Now, direct bio-transformation from GL to GAMG is becoming a hot spot. This paper studied the preparation and purification of GAMG. The main studies of this paper were as follows:
1) A new method of reversed-phase high performance liquid chromatography (RP-HPLC) was developed for the simultaneous determination of GL and GAMG in the enzymatic transformation system. The analysis adopted a fixed phase which was Apollo C18 column, a mobile phase which was V(methanol) : V(water) : V(acetic acid ) = 75:25:5 at a flow rate of 1.0 mL/min, and UV detection at 254 nm. The linear ranges of GL and GAMG were all at 0.2~20 μg. The recoveries of GL and GAMG were 97.47%~99.32% and 92.68%~98.44%, and the relative standard deviations were at the range of 0.88% ~3.78% and 1.07%~3.95% (n=3) respectively. The method is accurate, simple, rapid and cheap for simultaneous determination of GL and GAMG in the enzymatic transformation system.
2) Two kinds of preparation methods of rude GAMG were studied in this paper. On the one hand, bio-transformation of GL to GAMG used solid enzyme. After extraction by organic solvent and evaporation out of the solvent, rude GAMG was obtained; On the other hand, free state enzyme and membrane immobilization reaction assemblage were utilized to bio-transformation GL and preparation GAMG. After spray drying, rude GAMG was obtained.
3) The adsorption, desorption and isolation of GL and GAMG by resins were studied systematically. This part concluded follow aspects.
(1) Based on the different adsorption condition of nine resins, HP-20, NKA, X-5 macro-porous resins and D380 weak-base anion exchange resin were chosen to study that pH affected their adsorption affinity and capacity.
(2) D380 resin offered much higher adsorption capacity and affinity for GL and GAMG than other three resins, and NKA resin showed the worst adsorption affinity of the four resins based on calculated results from the measured adsorption isotherms. The adsorption data of X-5 on GAMG and D380 on GL fit the best to the Freundlich isotherm. Compared the isotherm of GL and GL in the GAMG solution, existence of GAMG would affect the adsorption of GL and form competive adsorption.
(3) Different concentrations of ethanol-aqueous solutions were used as elutions to study the static desorption followed the adsorption equilibrium. The results showed that ethanol-aqueous solutions did not have the selectivity for the elution of GL and GAMG from the HP-20, NKA and X-5 macro-porous resins, while they could not elute GL and GAMG from D380 anion exchange resin. Different concentrations of NaOH and ammonia aqueous, or different concentrations of NaCl and ethanol solution were added to above solutions were utilized as the elutions of D380 resin because its adsorption affinity was high and could not elute by ethanol. The experiments showed that the desorption ratio was low and there was no selectivity. So HP-20, NKA and X-5 macro-porous resins were selected to do the adsorption kinetics.
(4) Adsorption velocity of GL on NKA macro-porous resin was slower than HP-20 and X-5 macro-porous resins. Adsorption capacity of GL on NKA resin was relatively fewer than other two resins before the adsorption equilibrium. Adsorption kinetics curves of the three resins were fundamentally equal. Economic, adsorption coefficient of crossing over and above adsorption condition were considered. So NKA resin was chosen to do further experiments.
(5) Dynamic adsorption and desorption experiments have been carried out on a NKA resin packed column to separate GL and GAMG. Different concentrations of ethanol-aqueous solutions were used
as elutions eluted the column after dynamic声阻抗率
adsorption. When the concentration of ethanol-aqueous was over 70%, purified GAMG of 85.02% purity can be separated from crude enzymatic transformation in one column and the yield was 35.36%.
4) GAMG of the purity above 98% was prepared by preparative HPLC from GAMG of 85.02% purity, and it was determined by MS and NMR.
KEYWORDS: glycyrrhizic acid, glycyrrhetic acid 3-O-mono-β-D-glucuronide, macro-porous resins, adsorption, desorption
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