|关键词||分子印迹聚合物 菊苣酸 绿原酸 离子液体 在线固相萃取|
菊苣酸和绿原酸是存在于天然药用植物中的两种重要的酚类化合物，由于其具有较好的生理和药理活性，它们已经广泛应用于医药、保健品等很多行业。目前，分离和纯化这两种单体化合物的方法主要包括层析色谱、柱色谱和高速逆流色谱，但是，由于天然药用植物中成分比较复杂和结构类似物的存在,导致这些方法具有耗时长、溶剂消耗量大和操作繁琐等缺点。因而，迫切需要一种快速、简便和高效的方法从天然药用植物分离纯化主要化合物。本文运用原位聚合的方法制备菊苣酸和绿原酸分子印迹聚合物，并将聚合物作为固相萃取的吸附剂填料用于天然药用植物中这两种化合物的分离纯化。（1）以金属锌离子为枢纽制备分子印迹聚合物用于解决菊苣酸分子内氢键的方法，成功地从苦苣（Cichorium intybus L.）根粗提物中分离纯化出菊苣酸。分别以菊苣酸为模板，4-乙烯基吡啶为功能单体,醋酸锌为金属枢纽在离子液体中制备出菊苣酸印迹整体柱。色谱评价结果表明,通过比较加入和不加锌离子制备分子印迹聚合物的印迹因子,证明锌离子的加入有助于显著提高印迹聚合物对菊苣酸分子的印迹效应。当菊苣酸/Zn2+/4-乙烯基吡啶的摩尔比为1:2:24时, 所得到最高的印迹因子为 24.81。另外，将此条件下得到的菊苣酸印迹聚合物作为固相萃取柱的吸附剂填料并用于苦苣粗提物的分离，最优的淋洗和洗脱试剂为乙腈-甲酸-水（90:2:8, v/v/v）各10 mL，从苦苣粗提物中得到的菊苣酸的纯度和回收率分别为98.0% ± 0.5%和77.5% ± 2.5%（n = 6）。（2）离子液体诱导的分子印迹整体柱用于超快速在线分离和纯化。以绿原酸为模板，4-乙烯基吡啶为功能单体,二甲基丙烯酸乙二醇酯为交联剂,离子液体/二甲亚砜为致孔剂，偶氮二异丁腈为引发剂，混合后得到预聚混合物，超声后装入不锈钢柱中，密封水浴60 °C反应18 h后得到绿原酸印迹整体柱。最优化条件下所得到的整体柱对绿原酸最高的印迹因子为9.68。通过选择性实验可以看出，绿原酸分子印迹整体柱对绿原酸的保留作用强于其类似物（没食子酸甲酯，异槲皮苷，3-羟基苯甲酸，秦皮乙素，槲皮素和原儿茶酸），证明整体柱对绿原酸有很强的特异选择性。另外，离子液体诱导的印迹整体柱具有很好的通透性（1.53×10-13 m2）并允许高流速流过柱体，并将此绿原酸印迹整体柱用于超快速在线分离杜仲叶粗提物中的主要成分。当进样浓度和体积分别为6 mg mL-1和50 μL, 流速为5.0 mL min-1并在流动相为乙腈-乙酸钠缓冲盐（90:10, v/v）的条件下,得到高纯度的绿原酸且回收率为89.01% ± 0.05%（n=3）；另外，当粗提物进样量不超过50 μL时，可以获得另外一种化合物京尼平苷酸，其平均纯度和回收率分别为92.73% 和 67.72%。
Chicoric acid and chlorogenic acid are two significant phenolic compounds, which exist in natural medicinal plants. Because of their excellent physiological and pharmacological activities, they have been widely employed to many industries, such as medicine and health care and so on. To date, methods for separating and purifying the two compounds mainly involve with liquid chromatography, column chromatography, and high-speed countercurrent chromatography. However, these traditional methods are tedious, time- and solvent-consuming procedure, which are attributed to complex composition and the existence of structural analogy in natural medicinal plants. Therefore, a facile, convenient and efficient technique for separating and purifying of two compounds from natural medicinal plants is of urgent importance. In this paper, molecularly imprinted polymers are prepared by in-situ polymerization method, and the resulting polymers are applied as solid-phase extraction sorbent for the separation and purification of two compounds from natural medicinal plants.(1) A strategy of molecularly imprinted ploymers using zinc ion as the metallic center for solving the intramolecular hydrogen bonding of chicoric acid, which can successfully isolate and purify cichoric acid from extraction of Cichorium intybus L. roots. Molecularly imprinted polymers were prepared in a surrounding of RTILs, with cichoric acid, 4-vinylpyridine, and Zn(Ac)2 as template, functional monomer, and metallic pivot, respectively. The chromatographic evaluation showed that the presence of zinc ion can significantly improve the imprinting effect of the imprinted polymer by comparing the imprinting factor of the molecularly imprinted polymer synthesized with and without zinc ion. The highest imprinting factor was 24.81 when the molar ratio of chicoric acid/Zn2+/4-VP was 1:2:24. In addition, the chicoric acid-imprinted polymers were employed as an adsorbent for solid phase extraction, and used for the separation of chicoric acid from crude extracts of Cichorium intybus L. roots. The optimal washing and elution solution was acetonitrile-methanoic acid-water (90:2:8, v/v/v) (10 mL). Moreover, the purity and the recovery of chicoric acid from crude extracts of Cichorium intybus L. roots were 98.0% ± 0.5% and 77.5% ± 2.5% (n = 6), respectively.(2) The ionic liquid-induced molecularly imprinted monolithic column was used for ultra-fast on-line separation and purification. A pre-polymerization mixture was obtained utilizing chlorogenic acid, 1-butyl-3-methyl-imidazoliumtetrafluoroborate /dimethyl sulfoxide, 4-vinylpyridine, ethylene glycol dimethacrylate, and azobisisobutyronitrile as template, porogen, functional monomer, cross-linker, and initiator, respectively. And the mixture was injected into a stainless steel column after being sonicated. The chlorogenic acid imprinted monolith was obtained after the reaction was sealed in a water bath at 60 °C for 18 h. The highest imprinting factor was 9.68 under the optimal conditions. Selective experiment showed that the chlorogenic acid molecularly imprinted polymer had a stronger retention effect on chlorogenic acid than its analogs, and confirmed that the polymer had specific selectivity for chlorogenic acid. In addition, the imprinted polymer had excellent permeability (1.53×10-13 m2) and allowed an ultra-fast flow through the monolith. In addition, the chlorogenic acid imprinted polymers were employed for on-line separation and purification of the main components from E. ulmoides leaves extract. When the mobile phase was acetonitrile-sodium acetate buffer (90:10, v/v), and the flow rate was 5.0 mL min-1, 50 μL of extract from E. ulmoides leaves can be loaded on CGA-MIP monoliths and CGA with high purity can be obtained with recovery of 89.01% ± 0.05%. Furthermore, as the sample volume was below 50 μl, the mean purity and recovery of geniposidic acid were 92.73% and 67.72%, respectively.
|孙亚坤. 菊苣酸和绿原酸的分子印迹快速分离研究[D]. 北京. 中国科学院大学,2018.|
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