酶解水蛭地龙混合药渣的工艺研究及其产物的抗凝活性评价

Enzymatic hydrolysis of hirudo and pheretima residue and evaluation of anticoagulant activity of the product

关键词:  水蛭  地龙  酶解法  纤维蛋白原平板溶圈法  凝血酶滴定法

Key words:  hirudo  pheretima  enzymolysis  fibrinogen plate lysis  thrombin titration

摘要:

[目的] 考察蛋白酶酶解水蛭地龙混合药渣的工艺条件，评价其酶解产物的抗凝活性。[方法] 分别采用碱性蛋白酶、酸性蛋白酶和中性蛋白酶对混合药渣进行酶解实验。采用凝血酶滴定法和纤维蛋白原平板溶圈法评价酶解产物的体外抗凝活性，以确定最佳酶种类。溶液pH、酶底比、酶解时间、酶解温度为考察因素，单因素实验优化酶解工艺。[结果] 与药渣对照组和蛋白酶对照组对比发现，水蛭地龙混合药渣酶解液显示出较强抗凝活性。其中，碱性蛋白酶酶解产物的抗凝效果最好。其最佳工艺条件为酶底比0.95%、pH 11.4、酶解温度45 ℃，酶解时间4 h。[结论] 经碱性蛋白酶酶解后，水蛭地龙药渣酶解液的抗凝活性最强。实验为废弃药渣的再利用提供实验依据和开发方向。

Abstract:

[Objective] The technological conditions of enzymatic hydrolysis of hirudo and pheretima residue by protease were investigated,and the anticoagulant activity of the hydrolysate was evaluated.[Methods] Alkaline protease,acid protease and neutral protease were used to hydrolyze the mixed residues. Thrombin titration and fibrinogen plate lysis were used to evaluate the anticoagulant activity of the hydrolysates in vitro to determine the optimal enzyme type. The single factor experiment was carried out to optimize the enzymolysis process by selecting pH,enzyme substrate ratio,enzymolysis time and enzymolysis temperature as factors. [Results] Compared with the drug residue control group and protease control group,the mixture of hirudo and pheretima showed strong anticoagulant activity. Among them,alkaline protease hydrolysate had the best anticoagulant effect. The optimum conditions were as follows:enzyme substrate ratio 0.95%,pH 11.4,enzymolysis temperature 45℃,enzymolysis time 4 h. [Conclusion] The anticoagulant activity of the hydrolysate of hirudo and pheretima residue was the strongest after alkaline protease hydrolysis. This experiment provides experimental basis and development direction for the reuse of waste drug residue.