Baokang The Bupleurum Of 5s-of Rdna Intergenic Spacer Analysis And Quality Research

Root of Chinese Thorowax which is a main material of processed Chinese common traditional medicine has been used in China for more than 2000 years. It is the dried root of "Bupleurum chinense DC." and "Bupleurum scorzonerifolium Wild.", that are recorded in (2005 edition)《Pharmacopoeia of the People's Republic of China》,Root of Chinese Thorowax has a complicated origin in the practical application. The overwhelming majority of the plants of Genus Bupleurum (Umbelliferae) is used as the "Thorowax" at the local distribution areas, noxious Bupleurum longiradiatum is also without exception. And this is the main reason that the quality of the "Thorowax" and its preparation is not very steady.Presently the yield of "Thorowax" can't meet the increasing demands in the domestic and abroad market at present. In order to meet the needs of "Thorowax" markets, In recent years the Artificial Planted root of Chinese Thorowax has made great progress. "Baokang Thorowax" means "Thorowax" originating in Hubei Baokang, the actual species has been identified as Bupleurum chinense DC. Hubei Baokang is one of the main producing regions for "Thorowax", which not only has abundant wild resource but also has the special variety that own premium quality and yield. The Planting Area of "Thorowax" is increasing in recent years. A survey revealed that "Baokang Thorowax" confounded a small number of Bupleurum scorzonerifolium Wild. and Bupleurum marginatum. Implanting at liberty, unscientific management,inaccurate identification leading to the quality difference.Beginning with establishing a HPLC method for the determination of Saikosaponin a and Saikosaponin d in "Thorowax". Research on the quality judgment. Furthermore, genomic DNAs from "Thorowax", the sequences of 5s-rDNA spacer region were amplified and sequenced, and then analysed the genetic diversity with Mega3.0. Our work is significant for cultivation and quality identification in theory and practice.1. Determination of Saikosaponin a, d A HPLC method was established for the determination of Saikosaponin a and Saikosaponin d. Many parameters of HPLC technique was studied by a series of experiments. Finally a BonChrom C₁₈ column was used with acetonitrile-water (40:60) as the mobile phase and in a flow rate of 1.0 ml·min^-1 . The detecting wavelength was 204nm. The column temperature was 30℃.The average recovery rates were 99.29%, 99.44%, respectively while RSD were 0.73%,0.82%.Assay methods were reliable and accurate in the research of content. The experimental results indicate that: "Baokang Thorowax" better than "Henan Xiaodian Thorowax". "Thorowax" occured in Anjiawan, Dongjiagou and Bupleurum falcatum L. had similar content. Cultivated Thorowax better than the wild.2. Sequence divergence of 5s-rDNA spacer regionCommercial Kit methods was used to extract DNA from leaves. Many parameters of PCR were studied by a series of experiments, including DNA extraction, optimization PCR amplification by comparing a series of annealing temperature, template concentrations, amounts of Taq DNA polymerase and so on. The sequences were determined. Constructing phylogenetic trees with DNA sequences. The major results obtained from the experiment:The sequences of 5s-rDNA spacer region were similar. The length of them between 270 bp and 321bp,the G+C content is about 30.7～36.5%.We had the rescaled distance cluster combine and the proximity matrix, it showed that the relationship among the samples from different regions of "Thorowax". The correlation coefficient of them between 1 and 17.25. The genuine distance between species was larger than that within species, the genetic distance among genuine crude drugs was smaller. The main factor which affects the genetic distance maybe the different genetypes.It is also found that the content of Saikosaponin a, d is in accord with 5s-rDNA spacer region. The difference of Gene may cause genuineness. Correlation analysis of genuineness,gene and environment need more deep study.