水蛭酶解物抗凝活性肽类物质及作用机制研究

Whitmania pigra is a kind of traditional Chinese medicine with significant anticoagulant, antithrombotic effect. Previous studies have shown that the anticoagulant activity was greatly enhanced after Whitmania pigra was enzymolysised by trypsine. It’s supposed that some anticoagulant active peptides were released from protein with long chains. The anticoagulant mechanism were related to thrombin, fibrinogen and other factors. On the basis of former research, in this project, Whitmania pigra is enzymatic extracted firstly to obtain the enzymatic hydrolysate with best anticoagulant activity. And then the anticoagulant active peptides were purified through ion exchange, gel filtration, thrombin affinity chromatography and HPLC method. The structures of anticoagulant active peptides were analysized through MALDI-TOF, HPLC-MSn mass spectrometry. Finally, the direct interaction of enzymatic hydrolysate with thrombin and fibrinogen were studied through chromogenic substrate method and capillary electrophoresis. The indirect effect of enzymatic hydrolysate on thrombin and fibrinogen were studied through determining the content of thrombin and fibrinogen in plasma. In this project, anticoagulant active peptides were obtained through enzymatic extraction. It can provide a new idea for the reasonable and efficient use of Whitmania pigra. It also provides the basis for quality standard of Whitmania pigra. In addition, it can provide theoretical basis for the development of novel anticoagulant drugs.

水蛭是一味传统中药，具有显著的抗凝、抗血栓作用。前期研究表明，水蛭经胰蛋白酶酶解后抗凝活性大大增强，因此推测酶法提取可以将抗凝活性肽从长链的蛋白中释放出来，其抗凝作用机制除与凝血酶有关外，还与纤维蛋白原及其他凝血因子相关。在前期研究基础上，本项目首先对水蛭进行酶法提取，获得具有最佳抗凝活性的酶解物。然后利用离子交换、凝胶过滤、凝血酶亲和层析、HPLC等方法对水蛭酶解物进行分离纯化，获得抗凝血酶活性肽及其他抗凝活性肽，并结合MALDI-TOF、HPLC-MSn技术对抗凝活性肽进行结构分析。最后通过生色底物法和毛细管电泳法研究水蛭酶解物对凝血酶和纤维蛋白原的直接抑制作用，通过动物实验研究水蛭酶解物对血浆中凝血酶、纤维蛋白原含量的影响，阐明抗凝作用机制。该项目通过酶法提取从水蛭中获得抗凝活性肽，为水蛭的合理、高效利用提供新思路，也为水蛭的质量标准提供依据，同时为新型抗凝药物的开发提供理论基础.

水蛭是一味典型的具有抗凝活性成分的中药，其活性成分主要为蛋白多肽类成分，此类成分经消化道降解后吸收进入体内，因此体内发挥抗凝作用的成分应该是水蛭的降解产物。本项目以水蛭（宽体金线蛭）为研究对象，对其酶解物中的抗凝活性肽类进行研究并确定其抗凝作用机制。首先筛选出水蛭抗凝活性的测定指标—凝血酶时间（TT）法，然后使用该抗凝活性测定方法，采用正交设计法优化最佳的酶解工艺，最佳工艺为使用胰蛋白酶，温度73 ℃、酶底比10%、水解8 h，在此条件下可获得活性最好的水蛭酶解物；接着采用膜分离法、HPLC法对水蛭酶解物进行分离，并结合肠吸收实验，确定水蛭酶解物中的抗凝活性肽类成分为小分子量、大极性物质，经HPLC-MS鉴定出42个多肽和26个蛋白质。最后采用体内外实验对水蛭抗凝作用机制进行研究。生色底物法研究水蛭与凝血酶相互作用的结果显示水蛭在低浓度时(25mg/mL)具有诱导凝血酶活性作用，高浓度时（50mg/mL）具有抑制凝血酶活性的作用，抑制率为43%。SDS-PAGE法研究水蛭及其酶解物与纤维蛋白原的相互作用，结果显示水蛭提取液能够快速裂解纤维蛋白原的α、β、γ链，且裂解程度与水蛭浓度正相关。但水蛭酶解物与纤维蛋白原没有直接裂解作用。大鼠灌胃给予水蛭酶解物后测定血浆中的纤维蛋白原与凝血酶、凝血酶原，与空白组相比，结果显示大鼠灌胃给予水蛭酶解物后纤维蛋白原含量升高，凝血酶及凝血酶含量没有显著变化。因此水蛭不仅对凝血因子中的凝血酶有抑制作用，同时对纤维蛋白原也具有裂解作用。该研究结果对水蛭的合理用药提供了理论依据，同时明确了水蛭抗凝和溶栓的作用靶点。