Study On Q-Marker Of Schisandra Medicinal Materials

Objective:1.Through the combing and research of the medicinal literature of Schisandra chinensis,to formulate the standard of the medicinal materials of the "BeiWuwei".2.Through the study of the chemical constituents of S.chinensis,it provides a reference for the quality control and quality evaluation of S.chinensis,and establishes the chemical Q-Marker of S.chinensis.3.Through molecular identification,identify and analyze S.chinensis and related species,and find the biological Q-Marker unique to S.chinensis.4.Through the observation of the microstructure and powder of S.chinensis and Schisandra sphenanthera,the microscopic components of S.chinensis and S.sphenanthera were compared and analyzed to establish the morphological Q-Marker of Schisandra chinensi and Schisandra sphenanther.And draft a 2020 version of the Chinese Pharmacopoeia of the Schisandra sphenanther micro-identification standards.Materials and Method:1.Materials: 10 batches of S.chinensis collected in Fengcheng,Kuandian,Gaizhou,Jilin and Heilongjiang provinces from 2016 to 2017,12 batches of Schisandra sphenanther in Shangluo,Henan,Hubei,Shanxi,Shaanxi,and Liaoning from 2016 to 2017 More than 100 strains of S.chinensis were transplanted in Fengcheng,and more than 50 strains of Schisandra sphenanther were transplanted in Shangluo,Shaanxi Province.More than 20 Kadsura coccinea were transplanted from Huaihua City,Hunan Province,Conservation Nursery of key species of Medicinal plants of traditional Chinese Medicine in Liaoning Province(Liaoning,Dalian).2.Method:2.1 Investigate more than 50 historical documents on S.chinensis,and use the method of comparative analysis to sort out and sort out the medicinal history of S.chinensis.2.2 HPLC and UPLC-MS were used to analyze the chemical constituents of S.chinensis and S.sphenanthera.The qualitative analysis and quantitative analysis of six lignans in S.chinensis and S.sphenanthera,were compared to compare the content of six lignans in S.chinensis andS.sphenanthera.2.3 Molecular analysis method of S.chinensis and S.sphenanthera2.3.1 Using the Illumina Hiseq platform,High-throughput sequencing method was used to extract,amplify,purify,prepare,and sequence the total DNA of the mixed powder of S.chinensis and S.sphenanthera.Agilent Bioanalyzer was used to detect the data.Finally,the result of sequencing data was analyzed.2.3.2 Using the Illumina Hiseq platform,using the second-generation sequencing and three-generation sequencing technology,extract,collect,interrupt,build,and expand the chloroplast genomic DNA of fresh leaves of S.chinensis,S.sphenanthera,and Kadsura coccinea.Sequencing,and finally analyzing the subordinate data.2.3.3 The genomic Survey technique was used to evaluate the size,GC content and heterozygosity of the S.chinensis nuclear gene,and the experimental results were analyzed to provide a basis for genome-wide de novo sequencing.2.4 The tissue and powder of S.chinensis and S.sphenanthera(paraffin section)and powder(chloral hydrate and diluted glycerol)were studied by microscope according to the guiding principle of Pharmacopoeia technique,and observed,measured and photographed under microscope.Results:1.After combing and textual research,S.chinensis began to be published in?Shen Nong's Herbal Classic ?,as a top product.In the Ming Dynasty,? Original Materia Medica ?published "Liaowuwei bright red,long black",Li Shizhen first divided the north and south of S.chinensis,and pointed out that "the medicine for nourishing must be used by the northerners." The long-term observation of S.chinensis and S.sphenanthera has the phenomenon of "frost analysis",and the S.sphenanthera is more obvious.2.10 batches of S.chinensis and 12 batches of S.sphenanthera were determined by HPLC.The content of total lignan of S.chinensis was the highest in Fushun of Liaoning Province,which was 19.45 mg/g;.The average content of total lignan of S.chinensis was about 15 mg/g in Fengcheng and Huairen areas of Liaoning Province.The results of UPLC-MS provide a basis for the analysis of microchemical composition.3.The ITS2 sequences of mixed powder of S.chinensis and S.sphenanthera were analyzed by high-throughput sequencing.The results showed that 37601 ITS2 sequences were divided into196 OTU,sequences and the OTU representative sequences of S.chinensis and S.sphenanthera were successfully analyzed.Therefore,the medicinal materials of S.chinensis and S.sphenanthera were identified successfully.The chloroplast genomes of S.chinensis and S.sphenanthera and Kadsura coccinea were sequenced and their chloroplast genomes were146875 bp,146842 bp,145399 bp..The chloroplast genes were annotated and the genetic differences among species were compared.14 functional genes in chloroplasts were obtained,and the differences among them were found.The differences of length and distance between rps16 and petB and rpl16 and clpP were obtained by IR region boundary analysis.The information of nucleotide types and quantities of SSR could indicate the differences among the three species.We calculated the nuclear gene size of S.chinensis about 9 G,which laid a foundation for the de novo sequencing of S.chinensis nuclear gene.The results of the evaluation of Survey heterozygosity of the nuclear gene of S.chinensis were obtained.4.Through microscopic identification and analysis,we obtained that the common characteristics of S.chinensis and S.sphenanthera have the same structure of seed coat epidermis stone cells,seed skin inner layer stone cells,the difference is that the S.sphenanthera contains cluster crystal in the mesocarp cells,but not S.chinensis.The pericarp epidermis cells of S.chinensis were thickened in the shape of continuous beads,but the S.sphenanthera was not obvious.From these characteristics,we can identify both of them,and draw up a draft microscopic standard of S.sphenanthera in the 2020 edition of Chinese Pharmacopoeia?Chinese Pharmacopoeia?.Conclusion:1.This paper combs the medicinal history of S.chinensis and formulates the group standard draft of "Beiwuwei",which provides the basis for the good development of the cultivation and development of S.chinensis.It also provides a reference for the standard formulation of "Liaowuwei".2.Study on Chemical Q-Marker of S.chinensis(Schisandra L.)22 batches of S.chinensis and S.sphenanthera were determined,which provided theoretical data for the determination of thechemical Q-Marker of S.chinensis and S.sphenanthera and the quality evaluation of S.chinensis and S.sphenanthera.3.molecular identification conclusion3.1 The high throughput sequencing technique can accurately identify the powder of the mixture of S.chinensis and S.sphenanthera,which provides a new idea for the identification of the powder of mixed medicinal material.3.2 The chloroplast genome of S.sphenanthera was sequenced and analyzed for the first time,and the chloroplast genome of S.chinensis was uploaded to NCBI(SRX4282569).The chloroplast genome was successfully used to identify S.chinensis and related species,which provided reference for chloroplast as a super DNA bar code for identification and analysis of related medicinal plants.3.3 The nuclear gene Survey of S.chinensis was analyzed for the first time,and the gene size of S.chinensis was about 9 Gb,which provided a reference for the nuclear gene sequencing of S.chinensis.4.The microstructural analysis of S.chinensis and S.sphenanthera can be used to determine whether the epidermal cells of the pericarp have been ligated and whether they contain cluster crystals.The draft standard for microscopic identification of S.sphenanthera was developed for the foundation of 2020 version of the?Chinese Pharmacopoeia?.