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美洲大蠊抗肝纤维化活性部位酶解制备工艺的水解蛋白酶筛选
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| 篇名： | 美洲大蠊抗肝纤维化活性部位酶解制备工艺的水解蛋白酶筛选 |
| TITLE： | |
| 摘要： | 目的：优选美洲大蠊脱脂膏酶解工艺的最佳水解酶，以提高美洲大蠊中抗肝纤维化活性部位的提取得率和活性。方法：以美洲大蠊脱脂膏为对照，采用茚三酮法和福林酚法考察胰蛋白酶（TR）、胃蛋白酶（PE）、碱性蛋白酶（AL）、木瓜蛋白酶（PA）、中性蛋白酶（NE）对脱脂膏的水解度；采用大孔树脂分离纯化法，分别以50％、60％、70％、95％乙醇为洗脱溶剂，考察各酶解液不同溶剂洗脱部位的得率；借助大鼠肝星状细胞HSC-T6体外抑制试验，考察各酶解液洗脱部位的抗肝纤维化活性。结果：PA、NE对美洲大蠊脱脂膏的水解度分别为14.15％、15.70％，具有较强的酶解能力。PA、NE、AL酶解液的95％乙醇洗脱部位得率分别为（0.73±0.04）％、（0.65±0.01）％、（0.64±0.05）％，分别较不加酶的脱脂膏提高了30.36％、16.07％、14.29％。细胞体外抑制试验结果显示，各酶解液分离纯化所得的50％、60％、70％乙醇洗脱部位对HSC-T6细胞抑制率较低或呈现促增长现象；其95％乙醇洗脱部位对HSC-T6细胞的抑制率总体均在20％以上，其中PA、NE酶解液分离纯化所得的95％乙醇洗脱部位作用24～72 h时的半数抑制浓度（IC50）分别为94.5～112.3、117.1～120.0 μg/mL，总体小于不加酶的脱脂膏的IC50（116.1～123.0 μg/mL）。结论：综合水解度、纯化洗脱部位得率及对HSC-T6细胞的体外抑制活性3个指标，确定PA为美洲大蠊脱脂膏抗肝纤维化活性部位酶解工艺的最佳水解酶；NE、AL效果次之；PE、TR效果较差，不适于该酶解工艺。 |
| ABSTRACT： | OBJECTIVE： To optimize the proteolytic enzymes for enzymolysis technology of degreasing ointment from Periplaneta americana， and to improve the extraction rate and activity of anti-liver fibrosis active part from P. americana. METHODS： Using degreasing ointment of P. americana as control， ninhydrin method and folin-ciocalteu method were used to investigate the hydrolysis degree of trypsin （TR）， pepsin （PE）， alkaline protease （AL）， papain （PA） and neutral protease （NE） to the degreasing ointment. Macroporous resin isolation and purification method was used to investigate the yield of elution part from hydrolyzate， with 50％， 60％， 70％， 95％ ethanol as eluting solvents. Inhibition test in vitro of rat hepatic stellate cells HSC-T6 was performed， and anti-liver fibrosis activity of elution part from hydrolyzate was investigated. RESULTS： The hydrolysis degree of PA and NE were 14.15％ and 15.70％， showing strong enzymatic hydrolysis ability. The yield of 95％ ethanol elution part from PA， NE and AL hydrolyzate were （0.73±0.04）％，（0.65±0.01）％ and（0.64±0.05）％， improving 30.36％， 16.07％， 14.29％ compared with degreasing ointment without enzyme. Results of inhibition test in vitro showed that inhibitory rate of 50％， 60％， 70％ ethanol elution parts isolated and purified from hydrolyzate had a low inhibition rate or a growth-promoting effect on HSC-T6 cells. Inhibition rates of 95％ ethanol elution parts to HSC-T6 cells were all more than 20％. IC50 of 95％ ethanol elution part isolated and purified from PA and NE hydrolyzate for 24-72 h were 94.5-112.3 and 117.1-120.0 μg/mL， which were lower than that （116.1-123.0 μg/mL） of degreasing ointment without enzyme. CONCLUSIONS： PA is the best hydrolyzate for enzymolysis technology of active parts against liver fibrosis in degreasing ointment from P. americana， followed by NE and AL； PE and TR， which have poor effect， are not suitable for the enzymatic hydrolysis technology. |
| 期刊： | 2019年第30卷第14期 |
| 作者： | 杨华蕊，杨永寿，肖培云 |
| AUTHORS： | YANG Huarui，YANG Yongshou，XIAO Peiyun |
| 关键字： | 美洲大蠊；肝纤维化；水解蛋白酶；酶解工艺；水解度；分离纯化；HSC-T6细胞 |
| KEYWORDS： | Periplaneta americana；Liver fibrosis； Proteolytic enzymes； Enzymolysis technology； Hydrolysis degree； Isolation and purification； HSC-T6 cells |
| 阅读数： | 337 次 |
| 本月下载数： | 2 次 |
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