【作者】 罗玥佶；

【导师】 刘选明； 伍贤进；

【作者基本信息】 湖南大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 翻白草学名:Poterntilla discolor Bge,英文名:Discolor Cinquefoil Herb,蔷薇科委陵菜属多年生草本植物。具清热解毒、消炎镇痛、抗菌、抗病毒、降血糖等功能,在临床上多以翻白草的水煎剂用作内服药,如治疗出血性疾病,阿米巴痢疾等;或者结合其它中药水煎剂治疗风湿麻木瘫痪、筋骨久痛、风瘫、癫痫等。本文对翻白草的有效成分、药理作用和临床应用等方面的研究进行分析归纳,认为翻白草生物活性较高,疗效确切,值得进一步推广应用,应进一步加强翻白草的基础研究,研制现代中药新剂型。近年来翻白草的研究集中在其有效成分、药理作用等方面,还未见在分子水平上的研究。本文从分子水平初步研究了翻白草种质资源及属间药用植物的亲缘关系,同时建立了HPLC指纹图谱,对黄酮类化合物进行分析。主要研究内容有以下几个方面:翻白草种子具有休眠特性,自然条件下发芽率不高。本文采用赤霉素、碳酸钠、壳聚糖和低温层积处理研究解除翻白草种子休眠、促进萌发、提高发芽率的方法。本文利用ISSR（inter-simple sequence repeat）分子标记技术对翻白草同属的6种药用植物的亲缘关系进行了研究和分析。首先对翻白草ISSR体系进行建立和优化,确立了翻白草的ISSR-PCR的最佳体系。确立体系后,根据ISSR扩增结果发现了这种亲缘关系与地域分布有一定关系,从分子水平将供试材料区分开来,为DNA指纹图谱的建立奠定基础。同时对12份不同地区的翻白草种质进行了基因组DNA多态性分析。从50条引物中筛选出12条多态性引物用于PCR扩增,共扩增出128条DNA条带,其中多态性DNA条带104条,占总条带数的81.25%。根据ISSR扩增结果,利用POPGENE1.32软件计算了Nei’s基因距离,并用MEGA3.1软件通过UPMGA法进行了聚类分析。结果表明:翻白草种质资源具有较丰富的遗传多样性;ISSR技术可将12份供试的翻白草材料全部分开,根据ISSR遗传距离系数划分的类群与地理位置分布有一定关系。高效液相色谱（HPLC）是建立中药指纹图谱的一种重要方法。本文建立了翻白草及属间两种药用植物黄酮类化合物的HPLC指纹图谱。色谱条件为:色谱柱为Atlantis C₁₈（250mm×4.6mm,5μm）,流动相为甲醇:水=40:60,流速为1.0mL/min,柱温为25℃,检测波长:368nm,进样量20μL。结果检测出10个分离度良好的共有指纹峰,计算了共有峰的相对保留时间和相对峰面积,结果表明该方法可作为科学评价及有效控制中药质量的可行的定性方法。更多还原

【Abstract】 Poterntilla discolor Bge, the whole plant of Rosaceae Thunb., is a kind of traditional Chinese medicine. In current time, pharmacological experiments had proved that it had the function of antibacteria, anti-virus and diuresis, and could enhance body immunity. This thesis reviewed the research status of Poterntilla discolor Bge. The active ingredients、pharmacological action and clinical application were analyzed and summarized. The results indicated that its biological activities were high; its effects were positive and worth being further popularized, but basic research should be conducted to develop new herbal preparations.Recently, the research of Poterntilla discolor Bge have been focused on active ingredients、pharmacological action、planting technique and clinical application, but rarely on molecular level. The thesis studied the germplasm resources of Poterntilla discolor Bge elementarily, and the HPLC fingerprints had been constructed.Poterntilla discolor Bge seeds have dormancy property. It is difficult to break the dormancy during the natural conditions. In order to improve the dormancy-breaking process and the germination rate, this study investigated effect on the seed in dormancy-breaking with different methods such as low temperature and other chemical treatments.12 germplasm resources of Poterntilla discolor Bge were used as materials for analyzing their genome polymorphism by ISSR markers. 12 primers screened out from 50 primers were used for ISSR amplification. A total of 128 bands were generated, of which 104 bands were polymorphic bands (the percentage of polymorphic band, PPB=81.25%). Nei’similarity coefficients and genetic distances were calculated by using POPGENE software and the dendrogram were constructed by using UPGMA. The results indicated that there actually existed much genetic diversity on molecular level among the germplasm resources of Poterntilla discolor Bge, the genuieness of Poterntilla discolor Bge was related with the environments in some way. The relationship between genuieness、genetic and environmental factors of Poterntilla discolor Bge were discussed.HPLC fingerprints and a sensitive and specific method is an enssential way to study the herb fingerprint. The gradient elution mode was applied in chromatographic separation. Atlantis C18(250mm×4.6mm,5μm), flow rate: 1.0 mL/ min , detecting wavelength: 368 nm and the column temperature: 25℃.The samples collected from different areas were determined. The samples quality was assessed by Similarity Evaluation System for Chromatographic Fingerprint and this method had a good repeatability and reproducibility. The method can be used to identify and evaluate the quality of Poterntilla discolor Bge conveniently.更多还原