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目的优选蒙古黄芪Astragalus membranaceus mongholicus中2种免疫活性蛋白Am PR10-16k Da和HQGP-2的最佳提取工艺。方法以蒙古黄芪中所含可溶性蛋白Am PR10-16k Da和HQGP-2二级结构的圆二色性对提取温度和提取溶剂种类进行考察;对该2种蛋白提取条件进行单因素考察,并采用L9(34)正交试验设计法,采用Image凝胶图形分析软件以Am PR10-16k Da和HQGP-2在SDS-PAGE凝胶图中的蛋白条带灰度值(峰面积)为指标,考察提取温度、料液比、提取时间、提取溶剂(p H值)、药材粒度、提取次数对Am PR10-16k Da和HQGP-2蛋白条带灰度值的影响,从而确定Am PR10-16k Da和HQGP-2的最佳提取工艺,辅以其免疫抑制率(CCK-8法)和可溶性蛋白定量测定(BCA法)作为佐证。结果建立了蒙古黄芪中Am PR10-16k Da和HQGP-2的最佳提取工艺:向药材粉末(过4号筛)5.0 g中加入16倍量Tris-HCl,在温度40℃条件下恒温提取60 min,以100 r/min搅拌。蒙古黄芪蛋白质提取率为65 mg/g,且质量浓度为90μg/m L粗蛋白的免疫抑制率为90.90%。结论优化的提取工艺能正确反映Am PR10-16k Da和HQGP-2收率的最高相对量,为Am PR10-16k Da和HQGP-2的进一步研究提供了稳定、合理、可行的提取工艺。
Objective To optimize the extraction of two immunologically active proteins Am PR10-16k Da and HQGP-2 from Astragalus membranaceus mongholicus. Methods The extraction temperature and extraction solvent were investigated by the circular dichroism of the secondary structure of Am PR10-16k Da and HQGP-2 contained in Astragalus membranaceus. The extraction conditions of the two proteins were investigated by single factor L9 (34) Orthogonal design method, using Image gel analysis software to Am R10GP10-16k Da and HQGP-2 in the SDS-PAGE gel chart of protein bands gray value (peak area) as an indicator, Extraction temperature, extraction ratio, extraction time, extraction solvent (p H value), drug particle size and extraction times on the gray value of Am PR10-16k Da and HQGP-2 protein were determined to determine Am PR10-16k Da and HQGP-2 the best extraction process, supplemented by its immunosuppressive rate (CCK-8 method) and quantitative determination of soluble protein (BCA method) as evidence. Results The optimal extraction process of Am PR10-16k Da and HQGP-2 was established: 16-fold amount of Tris-HCl was added to 5.0 g of medicinal powder (No. 4 sieve), and the mixture was heated at a temperature of 40 ℃ for 60 min, stirring at 100 r / min. Mongolian milk protein extraction rate of 65 mg / g, and the mass concentration of 90μg / m L crude protein immunosuppression rate of 90.90%. Conclusion The optimized extraction process can correctly reflect the highest relative yield of Am PR10-16k Da and HQGP-2, providing a stable, reasonable and feasible extraction process for the further study of Am PR10-16k Da and HQGP-2.