基于靶酶－中空纤维离心超滤－液质联用技术的中药中酪氨酸酶抑制剂的筛选及活性评价研究

Screening of the bioactive compounds from natural complex system, such as traditional Chinese medicines (TCMs), is one of the highlighted research challenges in the field of drug development. In the present project, a novel screening approach based on target enzyme - hollow fiber centrifugal ultrafiltration (HFCF-UF) coupled with high-performance liquid chromatography and mass spectrometry (HPLC-MS) was proposed to be established and applied to screening and characterizing enzyme inhibitors from TCMs. Tyrosinase will be chose as the target with the Chinese herbs (Radix Angelicae Dahuricae and Radix Glehniae) as research objectives. After incubation of the enzyme and extracts, the enzyme-active compounds complexes are separated from the unbound small molecules by HFCF-UF, the obtained ultrafiltrate is introduced to the HPLC-MS for separation and characterization, the compounds with binding affinity to the enzyme will be indirectly identified and the relative binding degree will be obtained by calculation of the relative decrease of peak area calibrated by internal standard. The activity evaluation of tyrosinase inhibition of the affinity ingredients will be further conducted by molecular, cell biology and whole animal experiments. The proposed screening system has the characteristics of rapid, efficient, sensitive and suitable for complex system. The project not only can be realized to discover the lead compounds with tyrosinase inhibition from Radix Angelicae Dahuricae and Radix Glehniae, but also expected to be applied to screening inhibitors of other enzymes from TCMs.

如何从中药等复杂体系中快速、高效地筛选活性成分，是现代药物研究领域的重要挑战之一。本项目拟建立一种基于靶酶—中空纤维离心超滤—高效液相色谱质谱联用的靶向亲和筛选新方法，并应用于中药中酶抑制剂的快速筛选与鉴定。即选择酪氨酸酶为靶标酶，以白芷和北沙参中药为研究对象，将中药提取物与靶酶孵育后，经中空纤维离心超滤将靶酶—活性成分复合物与未结合成分分离得超滤液，将其导入液相色谱质谱系统进行分离与结构鉴定，通过对比提取液孵育前后图谱，以经内标校正的相对峰面积减少法间接辨识与靶标酶具有亲和作用的成分并获知相对亲和力。用分子、细胞生物学和整体动物实验，进一步评价亲和成分对酪氨酸酶的抑制活性。该筛选体系具有快速、高效、灵敏、适用于复杂体系的特点，本项目可实现从白芷和北沙参中筛选出具有酪氨酸酶抑制活性的先导化合物，并有望推广应用于中药中其他酶抑制剂的筛选。

如何从中药等复杂体系中快速、高效地筛选活性成分，是现代药物研究领域的重要挑战之一。本项目选择酪氨酸酶为靶标酶，以白芷和北沙参中药为研究对象，建立一种基于靶酶—中空纤维—高效液相色谱质谱联用的靶向亲和筛选新方法。即将中药提取物在中空纤维与靶酶孵育后，将靶酶—活性成分复合物与未结合成分分离，后将靶酶—活性成分复合物分离，将其导入液相色谱质谱系统进行分离与结构鉴定，通过对比提取液孵育前后图谱，以相对峰面积减少法间接辨识与靶标酶具有亲和作用的成分并获知相对亲和力。对筛选所得的三个香豆素活性成分进行了分子验证；斑马鱼活性研究表明与阳性药熊果苷相比，香豆素活性成分活性较弱，而且具有一定的毒性。此外，利用超滤-液质联用与分子对接技术对筛选所得木蝴蝶总黄酮提取物进行了酪氨酸酶抑制剂的筛选，发现7个具有酪氨酸抑制活性的成分，分别为木蝴蝶苷B （1），山奈酚-3-O-β-D-龙胆二糖苷（2），白杨素-7-O-β-D-龙胆二糖苷（3），木蝴蝶苷A（4），白杨素-7-O-β-D-D-葡萄糖醛酸（5），黄芩素（6），7-白杨素（7）。该项目所建筛选方法具有快速、高效、灵敏、适用于复杂体系的特点，可实现从中药等复杂体系筛选出具有酪氨酸酶抑制活性的先导化合物，并有望推广应用于中药中其他酶抑制剂的筛选。