Genetic Diversity And Quality Evaluation Studies On Traditional Chinese Medicine Of Paridis

Paris belonged to the family of Trilliaceae or Liliaceae, It had24species in total,19of them existed in China and the Qionglai Mountain was the distribution center of Paris in Sichuan Province. Pharmacological researches in recent years showed that plants of Paris had the physiological activities of hemostasis, bacteriostasis, relieving cough and asthma, anti-tumor, immunoregulation and so on, which makes them widely applied in clinical practice and increase continuously in medicinal demands. Currently, only Paris polyphylla var.yunnanensis and Paris polyphylla Smith var. chinensis were documented by Pharmacopoeia as medicinal plants. Since the period of " SARS", with the demand of Paris increasing sharply, wild resources as the main source of P. polyphylla var. yunnanensis and P. polyphylla var. chinensis was already endangered and accompanied by pseudo,defective medicine problem. Therefore, the study included two contents,one is genetic diversity of Paris by RSAP and Scot markers,the other is chemical fingerprint of Rhizoma Paridis by HPLC for internal quality evaluation. It had important and far-reaching significance to preserve its germplasm resources, provided the reference to ensure the safety.and had important significance to expand base of legal Paris.(1) Application of RSAP and Scot to reveal genetic diversity of Paris (Ⅰ) RSAP-MarekersResearch of genetic diversity on Paris resources growing in Sichuan was conducted based on RSAP molecular markers.18pairs of primer combinations were screened from the total45primer combinations, which were combined from10different RSAP primers. And then we built up the optimized RSAP-PCR system. To40tested materials, RSAP amplified347bands whose polymorphism ranged from82.927%and91.813%, and114.45bands for average polymorphism in the optimized condition of RSAP-PCR. The products were dealt by PAGE electrophoresis and band numerical statement, and next we divided the40tested materials into4classes by cluster analysis through NTsyspc2.10e. They are:①.polyphylla var.stenophylla (No3,11,12,17,18,20,22,24,30,31,40) and P.mairei (No21);②P. fargesii(No1,2,16,19,28,29,32,34,36), P. thibetica(No4,8,9,10)and P.axiparis var.axialis (No23,33);③P. polyphylla var.yunnanensis (No35) and P. delavayi var. petiolata (No6,25,26,38);④P. polyphylla Smith var. chinensis (No7,14,15,27,37,39) and P. delavayi vardelavayi (No5,13)。(Ⅱ) Scot Markers11primers, which could be amplified into clear bands with diversity, were screened from36Scot primers to be applied to Scot-PCR for40tested materials,and built up an optimized conditions of Scot-PCR. Under the optimized conditions of Scot-PCR, a total of127bands with the polymorphism from70.833%to90.909%were amplified, and the average polymorphism were48.15. The products were detected by2.5%Agarose gel electrophoresis and then we divided the40tested materials into4classes through NTsyspc2.10e cluster analysis. They are:①P. polyphylla var.stenophylla (No3,11,12,17,18,20,22,24,30,31,40);②P. fargesii (No1,2,16,19,28,29,32,34,36), P. delavayi var. petiolata(No6,25,26,38), P. delavayi var delavayi (No5,13) and P. polyphylla var. yunnanensis (No35);③P. thibetica(No4,8,9,10) and P.polyphylla Smith var. chinensis (No7,14,15,27,37,39);④P.axiparis var. axialis (No23,33) and P. mairei (No21)(III) Comparative analysis of two molecular markersRSAP molecular marker designed primers based on restriction enzyme cleavage site,its products detected by PAGE (6%) with high resolution; and Scot molecular marker designed primers the initiation codon, its PCR had both single, double primer characteristics, its products detected by2.5%agarose gel electrophoresis; although there were a smaller difference to the internal clustering, while a big difference to interspecific analysis clustering, this result was related to the characteristic difference of different molecular markers,also contributed to develop more gene resources of P. polyphylla var.yunnanensis and P. polyphylla var.chinensis with important genetic information.This study had important and far-reaching significance to preserve germplasm resources of Paris.(2) Quality evaluationThe chemical fingerprints of the materials were generated by HPLC(High Performance Liquid Chromatography). In our research,the contents of Polyphyllin Ⅰ,Ⅱ, Ⅲ,Ⅵ and Ⅶ was determined at the same time to17commercial materials and65materials of wild.(I) correlation analysis Correlation analysis of polyphyllin showed one of dioscin(polyphyllin Ⅰ,Ⅱ,Ⅲ) has significant correlation to the other(polyphyllin Ⅰ,Ⅱ,Ⅲ),While there was not clear to the relevance for pennogenin saponins (polyphyllin VI, VII). This study showed polyphyllin III maybe measure the medicinal quality of Paris.(Ⅱ) Contents for polyphyllinBased on the Chinese Pharmacopoeia(2010edition), only3copies from17commercial materials was detected to be qualified by the total contents of polyphyllin Ⅰ,Ⅱ, Ⅵ and Ⅶ only17.6%for the qualified;11copies of qualified and the rate of16.9%for the wild materials.ln our research.we also found that P. vietnamensis(SP5from mianyang city and SP18from ya’an) was qualified and this result showed P. vietnamensis had some medicinal value and certain alternative potential to legal Paris. In addition, According to related research we found optimum harvest period of Paris is not a fixed growth years, for which may be light, composition of the soil and water, humidity and other factors.(Ⅲ) The similarity for chemical fingerprintsWith SP6(was P. polyphylla var chinensis) to be control, we calculated the similarity to chromatographic fingerprints for the remaining17commercial materials, showed that commercial Paris may well existed quality problems with uneven. And through the comparison of similarity from to65wild from Ya’an,we held that P. polyphylla var stenophylla, P. delavayi var delavayi, Pmairei had a high homogeneity to P. polyphylla var chinensis. It had important significance to expand the legal basis of Paris judging from the overall and systematic of Traditional Chinese Medicine.