金莲花蛋白的鉴别特征及活性研究

【作者】 张丽娟；

【导师】 张贵君；

【作者基本信息】 北京中医药大学 ， 中药学， 2006， 硕士

【摘要】 中药金莲花为毛茛科植物金莲花(Trollius chinensis Bunge.)的干燥花。是一种具有较高抗菌能力的中药材,现代药理学研究证明:金莲花有抗菌、抗病毒等作用,广泛应用于治疗呼吸道和肠道感染等。以定量药理学和分子生物学技术为基础,能够客观地反映中药效价或信息物质(蛋白质和核酸等)特征等指标的“生物鉴定法”为评价中药质量的先进方法之一。本实验建立了金莲花蛋白的SDS-PAGE电泳实验体系,在蛋白质水平上对不同产地、不同采收时间的金莲花样品进行了研究,为金莲花的信息物质鉴定指标的建立提供相关信息。并对金莲花蛋白进行了初步抑菌活性实验,为金莲花的进一步开发利用提供了参考数据。本实验研究结果如下:1金莲花蛋白的提取在与其它提取方法比较的基础上,采用L9(34)正交设计优化超声提取金莲花蛋白。最佳超声提取条件为:原料粒度60目,料液比1:30,提取时间30min。2金莲花蛋白SDS-PAGE实验体系的建立电泳条件:分离胶浓度为12%;浓缩胶浓度为5%;样品上样量为20uL;电泳时间2.5h左右。通过比较几种文献提供的蛋白质凝胶染色方法及其染色结果确定适合本实验的最佳染色脱色方法,利用此方法染色洗脱,得到背景色浅,蛋白谱带清晰的凝胶电泳图谱。并初步尝试了梯度胶(5%～20%)凝胶电泳来分离样品蛋白质。结果表明:通过对SDS-PAGE凝胶电泳图谱的分析,9种金莲花样品均含有10～14条谱带,分别为约10KD、14KD、17KD、21KD、30KD、33KD、39KD、41KD、45KD、53KD、60KD、64KD、70KD、81KD。其中1～7号及9号样品的电泳谱带中,一级谱带4条(70KD、45KD、41KD、33KD),二级谱带4条(81KD、60KD、53KD、30KD),其余为三级谱带。但不同金莲花样品在蛋白谱带的级别、分布等方面差异显著,64KD在花蕾样品中(5,8号)表现为一级谱带,在其他样品中表现为三级谱带,可作为金莲花不同生长期的一条标志性谱带;70KD在2号样品(2002年采收)谱带颜色深浅接近二级谱带,比其他贮藏期短的样品色浅,表明贮藏期的长短对蛋白有影响。另外电泳谱带39KD在栽培品(1,9号)表现为二级谱带,而在野生样品中表现为三级谱带,表明野生与栽培品所含蛋白的含量存在差异。因此,通过分析电泳图谱中特征性谱带的级别、分布、条数的差异,可找出金莲花样品之间的鉴别特征,用于鉴定金莲花样品。更多还原

【Abstract】 Flos Trollii Chinensis is the dryness flower of Trollius chinensis Bunge. It is one of higher valuable antimicrobial Chinese traditional medicine. Modern pharmacology research proves: Flos Trollii Chinensis has widely antimicrobial spectrum. It has been widely used in treating respiratory and gastrointestinal infections.Based on quantitative pharmacology and molecular biology technologies, we can objectively reflect Chinese medicinal value or give out other indicators of genetic characteristics of "biological identification method" as one of the advanced methods for evaluating the quality of Chinese traditional medicines.The experiment established the Flos Trollii Chinensis protein electrophoresis testing SDS-PAGE system, studied in protein levels of different regions and different collection time samples, which will bring some information for further investigation of Flos Trollii Chinensis. The primitive anti-microbial activity of Flos Trollii Chinensis protein has been investigated, which will direct for the exploitation of Flos Trollii Chinensis.The main results in this experiment were shown as follows:1 The extraction of Flos Trollii Chinensis protein.Base on studying and comparing with other methods,the L9(34) orthogonal test has been used to optimize the extraction method. The optimized ultrasonic method was as follows: powdering 60 mu, adding 30 times water, extracting for 30 minutes.2 The establishment of the SDS-PAGE experimental system for Flos Trollii Chinensis protein.The experiment condition shows as: the separation gel’s concentration is 12%, the concentrate gel’s concentration is 5%, the sample’s quantity is 20μL, and the electrophoresis time last for about 2.5hours.By using the best dyeing method to treat the gel, and the clear protein strips with hypochromic background had been acquired. The gradient gel (5%-20%) has been attempted to separate the protein.The experiment analysis showed: By using SDS-PAGE system the total Flos Trollii Chinensis protein can be separated to 10-14 components, which are 10KD、14KD、17KD、21KD、30KD、33KD、39KD、41KD、45KD、53KD、60KD、64KD、70KD、81KD. Among them, the sample 1 to 7 and sample 9 have 4 primal grade bands(70KD、45KD、41KD、33KD), and 4 secondary grade更多还原