[关键词]
[摘要]
目的 研究中药牛骨髓中蛋白、多肽类成分的提取方法、结构信息及其抗菌活性,提高药用价值。方法 采用10种提取和3种制备方法,以蛋白含量、提取率、水解度、抗菌活性和相对分子质量等为考察指标,筛选牛骨髓蛋白与多肽的最佳提取和制备部位方法。结果 水、NaCl溶液和Tris-HCl溶液提取法得到的蛋白综合指标较高,其蛋白量分别为(52.20±0.58)、(50.19±0.21)、(43.21±0.05)mg/mL,抗菌活性的强弱与蛋白量高低有一定的关系;电泳图谱显示,牛骨髓蛋白样品具有2级谱带,分别为蛋白谱带(66 000)与多肽谱带(4 100~10 000);LC/MS数据显示,水提、0.5 mol/L NaCl和30%(NH4)2SO4部位多肽相对分子质量分别为1 027.84~9 916.32、1 131.17~4 989.95、1 150.46~9 917.00,与其电泳谱带基本吻合。结论 水提法操作较简单、快速、成本低,适合于工业化生产;采用SDS-PAGE和LC/MS对牛骨髓蛋白进行分析,为构建其指纹图谱提供理论依据。
[Key word]
[Abstract]
Objective To study the extraction method, structure information, and antimicrobial activity of the protein and polypeptide components from Chinese medicine bovine bone marrow (BBM) for improvement of its medicinal value. Methods Ten kinds of extraction and three kinds of preparation methods were used to take the protein content, extraction rate, degree of hydrolysis, antimicrobial activity, and molecular weight as investigation index to screen optimal extraction ability and preparation parts of bovine bone marrow protein (BBMP) and polypeptide. Results The comprehensive evaluation index of protein extracted in water, NaCl, and Tris-HCl solutions was high, their contents were (52.20 ±0.58), (50.19 ±0.21), and (43.21 ±0.05) mg/mL, respectively. The antimicrobial ability was related to protein content. The SDS-PAGE analysis showed that the proteins from BBM contained approximately secondary subunits which related to protein and peptide with molecular weight in the range of 66 000 and 4 100-10 000. The LC/MS data showed that the molecular weight of peptide extracted in water, 0.5 mol/L sodium chloride, and 30% (NH4)2SO4 solutions were 1 027.84-9 916.32, 1 131.17-4 989.95, and 1 150.46-9 917.00, respectively, and the results were basically consistent with 1-DE electrophoresis bands. Conclusion Water extraction method is simple, and fast with relatively low cost, which is suitable for industrial production of BBMP. The SDS-PAGE and LC/MS method is adopted to analyze and provide a theoretical basis for construction of BBMP fingerprint.
[中图分类号]
[基金项目]
国家科技重大专项—重大新药创制—民族药新品种研发及其关键创新技术(2017ZX09301045);新疆医科大学与中国科学院新疆理化技术研究所技术服务项目(20171010)