半夏总蛋白提取及其动态变化研究

目的优选出半夏总蛋白提取的最适方法并研究不同生长时期总蛋白量及成分的变化,为半夏药用成分的监控及合理采收提供科学依据。方法采用丙酮沉淀法、95%(NH4)2SO4盐析法、TCA-丙酮法和Tris-饱和酚法提取半夏总蛋白,筛选半夏总蛋白提取的最佳方法。分别于半夏出芽期、全苗期、珠芽期、佛焰苞期及倒苗期采集样品,分为新鲜组和干燥处理组,提取样品总蛋白,进行SDS-PAGE电泳检测及Bradford浓度测定。结果丙酮沉淀法、95%(NH4)2SO4盐析法、TCA-丙酮法和Tris-饱和酚法提取的半夏总蛋白量分别为0.369、0.678、1.082、0.493 mg/mL;新鲜半夏5个不同生长时期总蛋白量占块茎的质量分数分别为出芽期4.53 mg/g、全苗期2.28 mg/g、珠芽期2.79 mg/g、佛焰苞期7.61 mg/g、倒苗期10.21 mg/g;干燥半夏5个不同生长时期总蛋白量占块茎的质量分数分别为出芽期31.85 mg/g、全苗期18.52 mg/g、珠芽期42.08 mg/g、佛焰苞期28.56 mg/g、倒苗期56.84 mg/g;新鲜半夏块茎中蛋白含有15条蛋白带,干燥处理半夏块茎中蛋白含有5条蛋白带。结论 TCA-丙酮法是半夏总蛋白提取的最佳方法,不同生长时期的半夏块茎中蛋白成分各异,倒苗期总蛋白量最高。

Extraction of total protein in tubers of Pinellia ternata and its dynamic change

Objective To select the best method for extracting total protein from the tubers of Pinellia ternata,study the dynamic change of total protein and components in different growing periods,and provide the information for medicinal ingredients monitoring and reasonable harvesting.Methods Four methods,acetone precipitation,95%(NH4)2SO4 salting,TCA-acetone,and Tris-saturated phenol methods,were used to extract the total protein from the tubers of P.ternata and optimize the extraction method.Samples were collected at different growing stages: bud stage,seedling stage,bulbil stage,spathe stage,and maturation stage,and divided into two groups,dry and fresh.Total protein was extracted,detected by SDS-PAGE electrophoresis,and determined by Bradford.Results The concentration of pinellia total protein extracted by acetone precipitation,95%(NH4)2SO4salting,TCA-acetone,and Tris-saturated phenol methods was 0.369,0.678,1.082,and 0.493 mg/mL,respectively.The mass fractions of the total protein in fresh pinellia tuber were 4.53 mg/g at bud stage,2.28 mg/g at seedling stage,2.79 mg/g at bulbil stage,7.61 mg/g at spathe stage,and 10.21 mg/g at maturation stage.However,the mass fractions of the total protein in dry pinellia tuber were 31.85,42.08,28.56,and 56.84 mg/g at the corresponding stages above.Fifteen protein bands were found in fresh pinellia tuber while five in dry pinellia tuber.Conclusion TCA-acetone precipitation is the most suitable method for total protein extraction from the tubers of P.ternata.Total protein components and content in pinellia tuber change in different growing periods.