Research On The Resource Distribution Of Pulsatilla Miller And The Quality Evaluation Of Pulsatilla Miller In China

Purpose:1.The classification of Pulsatilla chinensis(Bunge)Regel has always been a hot topic of related scholars.P.chinensis was first classified as Anemone L.,and then it was divided into P.Miller.At present,the Baitouweng mainly rely on wild resources,and the cultivation techniques are very limited.Under the trend of increasing demand for medicinal materials in successive years,we are facing the dilemma of insufficient supply of Baitouweng.Therefore,it is necessary to make a systematic exposition on the distribution of resources and the classification of P.chinensis,and explore the seed germination of P.chinensis,so as to lay a foundation for the development of cultivation and breeding technology of P.chinensis.2.Due to the shortage of authentic Baitouweng resources,and the traditional identification methods such as appearance and morphological characteristics,it is difficult to accurately identify Baitouweng and its counterfeits,so the quality evaluation and identification of Baitouweng is very urgent.Using HPLC fingerprints and molecular biology techniques,it provides an effective method for the quality evaluation and accurate identification of Baitouweng.Material and method:1.Materials:14 seeds,6 fresh leaves,94 medicinal materials and 10 aerial parts of P.chinensis were collected from the preservation nursery for key species of medicinal plants of Liaoning Province,Zhumadian city of Henan Province,Qianshan of Anshan city of Liaoning Province,Fengcheng city of Liaoning Province,Yichun city of Heilongjiang Province and Tongliao city of Inner Mongolia Autonomous Region,respectively.They were identified by Professor Kang Tingguo and Professor Xu Liang of Liaoning University of traditional Chinese medicine.2.Methods:2.1 The P.Miller in China was systematically classified by referring to Flora Reipublicae Popularis Sinicae,Flora of China and related literature.The regional distribution of P.Miller resources in China was sorted out by consulting related literature.At the same time,according to the collection of P.Miller specimens in seven large herbarium of different regions in China,and the number and species of P.Miller specimens collected were sorted out.2.2 The 1000 seed weight of P.Miller was calculated and the seed germination experiment was carried out.2.3 With reference to the 2020 edition of Chinese Pharmacopoeia and related literature,the content of the index component Pulsatilla saponins B₄in 60 batches of three kinds of Baitouweng of P.Miller was determined by HPLC.First,the three medicinal materials of Baitouweng,Chaoxian Baitouweng,and Xingan Baitouweng are divided into large and small groups,and their length,diameter,and weight are measured,and the contents of different medicinal materials and the same medicinal materials in different groups are compared.2.4 HPLC fingerprints of 30 batches of Baitouweng and 10 batches of its aerial parts were established.Through the similarity evaluation system of TCM chromatographic fingerprints,the similarity of the obtained fingerprints was calculated,and the median method was used to generate the control fingerprints.The known chemical components were compared,and the retention time and peak area ratio of common peaks were calculated.2.5 The chloroplast genomes of six species of P.Miller were sequenced,assembled and annotated by Illumina hiseq sequencing platform,and their genomic characteristics,simple repeats,large repeats,IR region boundaries and nucleotide diversity were compared and analyzed.The phylogenetic tree based on chloroplast genome and 51 shared protein codes was constructed by neighborhood-joining method(NJ),and the phylogenetic position of P.Miller in Ranunculaceae was elucidated at the molecular level.2.6 High-throughput sequencing technology was used to identify four kinds of Baitouweng with different proportions of mixed powder.The sample proportions were 1:1:1:1,4:1:1:1,8:1:1:1,16:1:1:1.Using metagenomics strategy for analysis,using MEGA 6.0 to compare the ITS2 sequences of P.Miller selected from the NCBI database,and the selected OTU representative sequences were analyzed and identified.Through abundance analysis,explore the correlation between the amount of each sample sequenced and the number of sequences obtained.Results:1.By consulting the related literature,Linnaeus first attributed the P.chinensis to the Anemone L.in Species Plantarum in 1753 and named it Anemone pulsatilla.Nowadays,P.chinensis is classified as P.Miller.In the literature published in 1922,some scholars believe that P.chinensis var.kissii is a hybrid of P.chinensis and P.cernua.As a variety of P.chinensis,P.chinensis var.kissii is recorded in Flora Reipublicae Popularis Sinicae.There are 11 species,6 forms,2 subspecies and 5 varieties of P.Miller in China,totaling 24 species.2.Through the collection and collation of literature,it is found that P.Miller are distributed in20 provinces of China.As the only legal source of authentic medicinal materials,Baitouweng is widely distributed in Liaoning,Jilin,Heilongjiang,Hebei and other 16 provinces in China,and the most widely distributed in Northeast China.There are 11 species,3 varieties and 2forms of Chinese P.Miller in 7 large herbarium of different regions in China.There are 27species,4 subspecies,5 varieties and 7 forms of foreign P.Miller in 7 Herbarium.3.The appearance and morphology of the seeds of P.Miller are similar,but there are still slight differences in seed size and the length of persistent style.Seed germination experiment showed that the germination cycle of P.chinensis,P.cernua,P.dahurica,P.cernua f.plumbea,P.chinensis var.kissii and P.turczaninovii were 25 days,23 days,26 days,26 days,25 days and 24 days respectively.The germination rate was 31.33%,78.67%,74.00%,75.33%,32.00%and 36.00%respectively,and the germination potential was 23.33%,30.00%,56.00%,42.00%,24.00%and 32.00%respectively.The old seeds of P.chinensis and P.cernua did not germinate.4.HPLC method was used to determine the content of Pulsatilla Saponin B4 in 60 batches of samples from three kinds of Baitouweng.The average content of Pulsatilla saponin B₄in the large group of Baitouweng is 6.223%,and the average content of Pulsatilla saponin B₄in the small group of Baitouweng is 5.189%.The highest content of Pulsatilla saponins B₄in Baitouweng is 8.42%,and the lowest content is 2.88%.The content of Pulsatilla Saponin B₄in Chaoxian Baitouweng and Xingan baitouweng was very low,which did not meet the requirements of Chinese Pharmacopoeia.5.The HPLC fingerprints of 3 kinds of Baitouweng and 10 batches of samples from aerial parts of P.chinensis were established.The results showed that there were 21 common peaks in Baitouweng samples,25 common peaks in Chaoxian Baitouweng samples and 17 common peaks in Xingan Baitoueng samples.There are 9 common peaks in three kinds of Baitouweng.The chemical constituents of P.chinensis and P.cernua were identified as Pulsatilla Saponin E,Pulsatilla Saponin H,Pulsatilla Saponin B₄,Pulsatilla Saponin E₃and Pulsatilla Saponin E₁.Xingan Baitouweng is similar to both,but Pulsatilla Saponin E₁has not been identified.The fingerprint peaks of the aerial part of P.chinensis are quite different from those of Baitouweng.6.The chloroplast genome of six species of P.chinensis was sequenced and compared.The results showed that they had similar genome size,but different from Aconitum carmichaelii and Coptis chinensis.There are certain differences in the size of chloroplast genes rpl16 and rps12.Among them,P.dahurica contains six kinds of nucleotides,and other five species of P.Miller contain five kinds of nucleotides except hexalnucleotide.In the large repeat analysis,49,49,38,38,49 and 49 pairs of large repeats were found in the chloroplast genome of six species of P.Miller.In addition,their IR regions are very similar,but they are quite different from those of A.carmichaelii and C.chinensis.The nucleotide diversity analysis of chloroplast genome showed that there were 108 coding genes and 105 non-coding genes.The rpl36 gene and ccs A-ndh D region could be used as two significant variation sites for the development of molecular markers.The NJ phylogenetic tree was constructed by using chloroplast genome and 51 shared protein coding genes.P.Miller were clustered into one branch,which better reflected their phylogenetic position in Ranunculaceae.7.Based on the high-throughput sequencing technology,391,381 effective sequences were obtained from the identification of four kinds of P.Miller with different proportions of mixed powder.Among them,266,851 sequences with 363 bp were the most.It can be seen from Gra Phl An's sample overall classification hierarchy tree that the relative abundance of P.Miller is very large and belongs to the dominant group.In the analysis of relative abundance of species at the family and genera level,it can be seen that the relative abundance of Ranunculaceae and P.Miller is much greater than that of other families and genera.At the species level,it can be seen that the relative abundance of Baitouweng is the largest among all species,followed by the Chaoxian Baitouweng.Conclusion:1.Through literature review,we have a systematic understanding of the classification of P.Miller in China.P.chinensis is distributed in many provinces throughout the country,Northeast China as the main production area has a large number of distribution.Nevertheless,the supply of genuine Baitouweng is still in short supply.In this case,it is suggested to strengthen the resource management of Baitouweng,and vigorously develop the cultivation of genuine Baitouweng,so as to reduce the phenomenon of insufficient supply of medicinal materials and abuse of counterfeit medicinal materials.2.Through the investigation and data collection in the Herbarium,it is found that the Herbarium of Institute of Botany,Chinese Academy of Sciences has the most species and quantity of P.Miller.The Herbarium of Wuhan Botanical Garden of Chinese Academy of Sciences collected only one sample of P.Miller,while the Herbarium of Wuhan Botanical Garden of Chinese Academy of Sciences and the Herbarium of Northwest A&F University did not collect any foreign samples of P.Miller.These information can provide practical significance for the classification and resource distribution of P.Miller.3.The appearance of the seeds of P.Miller is very similar,with only slight differences.The seed individuals of P.turczaninovii and P.chinensis var.kissii were relatively large,which was consistent with the description of morphological characteristics.The seed germination experiment showed that the old seeds of P.chinensis and P.cernua did not germinate,which proved that the seeds of the second year lost their vitality completely.This experiment provides a reference for seed germination and breeding of P.chinensis.4.The content of Pulsatilla Saponin B₄in three kinds of Baitouweng was determined by HPLC.The results of the experiment verified the quality evaluation of Baitouweng in Chinese Materia Medica in terms of the relationship between medicinal properties and the content of index components,which is better to be thick and long with solid quality.From the point of view of the content of Pulsatilla saponins B₄,which is an indicator component,Chaoxian Baitouweng and Xingan baitouweng can not be used as a substitute for authentic Baitouweng.5.By constructing an HPLC fingerprint,the number of chromatographic peaks detected by Baitouweng and Chaoxian Baitouweng is more and more similar,and the two may contain more similar saponins.There are some differences in the fingerprints of the three kinds of Baitouweng and aerial parts,which can provide an effective method for their quality evaluation and counterfeit identification.6.The chloroplast genomes of six species of P.Miller were sequenced by Illumina hiseq sequencing platform.The application of super bar code technology can find out the differences of six species of P.Miller at the molecular level,which can be used for the identification of them.The construction of NJ phylogenetic tree showed the evolutionary position of P.Miller in Ranunculaceae,which provided a reference for further study on the evolutionary history of P.Miller.7.ITS2 sequence can be used to identify the related medicinal materials of P.Miller.The relative abundance of Xiye Baitouweng and Xingan Baitouweng is positively correlated with the amount of sequencing samples.Although the relative abundance of Baitouweng and Chaoxian Baitouweng is very large,no correlation is shown.This experiment can provide an effective method for the identification of medicinal materials of P.Miller.It also provides a new idea for the identification of mixed powder of medicinal materials.