蒙古黄芪中2种具有免疫活性的可溶性粗蛋白提取工艺优选

Optimization of extraction technology of two immune active proteins from Astragalus membranaceus var. mongholicus

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 technology of immune active glycoproteins AmPR10-16kD and HQGP-2 from Astragalus membranaceus var. mongholicus （AMM）. Methods The optimized extraction temperature conditions were investigated by circular dichroism of water-soluble protein involving in AmPR10-16kD and HQGP-2 with secondary structure from AMM. The optimized extraction technology was investigated using single factor test and orthogonal test with gray value of water-soluble protein AmPR10-16kD and HQGP-2 as the index which was determined by Image of gel graphical analysis software. In this study, the effects of temperature, solid-liquid ratio, time, solvent, granularity, and times on gray value were investigated, for which the inhibitory effect of water-soluble protein was determined as an evidence by CCK-8 method, and the content of water-soluble protein is determined as an evidence by BCA method. Results The optimized extraction technique for proteins AmPR10-16kD and HQGP-2 in AMM was established, that was 5.0 g powder of AMM over the No.4 sieve, olvent Tris-HC1, solid-liquid ratio 1：16 and 60 min for extraction at the temperature of 40 ℃ and being mixed under 100 r/min. The water-soluble protein extract rate in the orthogonal test analysis was 65 mg/g, of which inhibitory effect was 90.90% at a concentration of 90 μg/mL. Conclusion The optimal extraction conditions could accurately reflect the relative amounts of AmPR10-16kD and HQGP-2 maximum extraction rate, providing a stable, reasonable, and feasible extraction process for further study of the bioactive substance of AMM.