Study On Three Standards Of Pinellia Ternate And The Correlation Between Genetic Material And Quality Of Different Leaf Types

Pinellia Rhizoma,the dry tubers of Pinellia ternata(Thunb.)Breit,was firstly recorded in the Shen Nong's Herbal Classic.The main producing areas of Pinellia Rhizoma are Gansu,Sichuan,Guizhou,Hubei,Shanxi,Hebei and other places.With the effects of dryness and phlegm,lowering vomiting,eliminating phlegm,it was widely applied in clinical compatibility and proprietary Chinese medicine formulas,such as Baohe Pills,Baoning Pinellia Granules,Pinellia Cough Syrup,and Huoxiang Zhengqi Liquid.This paper was mainly targeting at some prominent problems,including the insufficiency of the quality evaluation of Pinellia Rhizoma,a large amount of fakes on maket and the serious phenomenon of adulteration,the lack of scientific evaluation index of Pinellia grading,the unclear differences of Pinellia ternata genes and the intrinsic quality of different leaf types,and the lack of quality standards of stems for seeding.Taking the quality evaluation of medicinal materials as the core,we started the research about the improvement of quality standard,authenticity identification,the grading of medicinal materials,the genetic differences and quality association of different leaf types,and classification of Pinellia Rhizoma for seeding,all of which provide a scientific basis for the comprehensive quality improvement of Pinellia Rhizoma.The main findings were as follows: 1.Study on quality standard upgrading of Pinelliae Rhizoma:The traits,safety evaluation,content determination,fingerprint and supplementary test method of counterfeits were studied.The results showed that:(1)Pinelliae Rhizoma with a diameter range of 0.5~1.0cm was an important component.There was no significant difference in the extract,organic acid content and nucleoside content of Pinelliae Rhizoma in different diameter ranges.It was advised that the diameter of the medicinal materials be revised from "1.0~1.5cm" to "0.5~1.5cm".(2)For the detection of exogenous harmful substances such as heavy metals and harmful elements,pesticide residues and aflatoxins in Pinellia Rhizoma,only three batches of Pinellia Rhizoma have excessive levels of cadmium(0.015~0.039 ppm higher than the limit standard of 0.3 ppm),and the amount was relatively small,indicating that Pinellia Rhizoma was safe in terms of exogenous harmful substances.(3)Using HPLC,HPLC-MS,NMR and other methods,three new components such as trans-aconitic acid,cis-aconitic acid and fumaric acid were newly isolated and identified from Pinellia Rhizoma and the multi-component and multi-index content determination methods were established for the first time,which can simultaneously measure the contents of seven organic acid components or nine nucleoside components.It was recommended to include the content determination methods of oxalic acid,L-malic acid and citric acid into the Chinese Pharmacopoeia 2020 version.(4)Based on the determination of organic acids in Pinellia ternata,a fingerprint of organic acids in Pinellia ternata was established.Seven chromatographic peaks were identified,which can be used for quality control of Pinellia ternata.(5)A systematic study on the serious problem of adulteration with Pinelliae Pedatisectae Rhizoma in Pinellia Rhizoma was carried out.For the first time,the characteristic component triglochinic acid was isolated and identified from the Pinelliae Pedatisectae Rhizoma.Based on the characteristic component triglochinic acid,the supplementary test standard for Pinellia Rhizoma and decoction pieces was established,which had been approved by experts organized by the China Food and Drug Control Research Institute.The method has strong specificity and high accuracy,and can be used to detect the adulteration of Pinelliae Pedatisectae Rhizoma in Pinellia Rhizoma and decoction pieces.It was found that the total counterfeit rate was 47.1% through the detection of 123 batches of Pinellia Rhizoma and decoction pieces,among which the fake rate of unprocessed Pinellia Rhizoma was 69.2%,and the rate of counterfeit of Pinellia Rhizoma praeparatumcum zingibere et alumine was 30.8%,the rate of counterfeit in the Pinellia Rhizoma praeparatum cum alumine was 31.8%,and the rate of counterfeit in the Pinellia Rhizoma praeparatum was 44.4%.2.Research on the commodity grade of Pinellia RhizomaThe grading of traditional Pinellia Rhizoma was mainly based on the diameter and the number of grains per kilogram as the grading index.The diameter,extracts,contents of 7 organic acids and 9 nucleosides,and fingerprint of Pinellia Rhizoma with different diameters were analyzed.The results showed that:(1)There was no significant difference in Pinellia Rhizoma with different diameters,including extracts,the content of organic acid and nucleoside,but the overall trend was that the larger the diameter,the higher the content,but comparing the content of Pinellia Rhizoma from the same batch,it was irregularity.(2)Took the Pinellia Rhizoma with a diameter of 1.2~1.5cm as reference of fingerprint,the similarity was high in the Pinellia Rhizoma with a diameter of 0.8~1.0cm and 1.0~1.2cm,but there were differences in the Pinellia Rhizoma with a diameter of 0.5~0.8cm and 1.5~2.0cm,the similarity of which were less than 0.9.(3)The number of grains per kilogram of Pinellia Rhizoma with a diameter of 0.5~0.8cm was 3395~6020,with an average of 5108±675.The traits of Pinellia Rhizoma with diameter more than 1.5cm were severely mutated,with many subtubers alongside,and frequently mixed with Pinelliae Pedatisectae Rhizoma,and its proportion and batches in the Pinellia Rhizoma samples were small,not representative,it was recommended not to be included in the grading of the medicinal materials.Therefore,according to the three-level classification principle,the grading standard of Pinellia Rhizoma was mainly determined by the diameter of the medicinal material and the number of grains per kilogram.The first grade of Pinellia Rhizoma: 1.2 cm < diameter ? 1.5 cm,within 1000 capsules per kilogram;the second grade of Pinellia Rhizoma: 1.0 cm <diameter ? 1.2 cm,less than 1800 capsules per kilogram;the third grade of Pinellia Rhizoma: 0.8 cm < diameter ? 1.0 cm,less than 3200 capsules per kilogram.3.Seed-stem grading standards of Pinellia RhizomaThrough analys was on the quality index of seed-stem,field test,yield and quality of medicinal material,the grading standards of seed-stem were established.Among them,the first grade seed-stem :0.5 <diameter ? 1.0cm,the purity ? 97%,the clarity ? 90%,moisture ? 50%,germination rate ? 95%;the secondary grade seed-stem: 1.0 < diameter ? 1.5cm,the purity ? 97%,the clarity ? 90%,moisture ? 50%,germination rate ? 90%.4.Correlation between intrinsic inheritance and quality of different leaf types of Pinellia ternata(1)Analysis of chemical constituents of different leaf types of Pinellia ternata There were significant differences in total alkaloids and total organic acids in different leaf types of Pinellia ternat.The contents of total alkaloids and total organic acids were the highest in bamboo leaf type,followed by peony and peach leaf type,and the lowest was in willow leaf type of Pinellia ternata.There were also some differences in the content of seven organic acids in different leaf types of Pinellia ternata,all of which mainly contain citric acid.Among them,the content of tannic acid in Pinellia ternata with peach leaf was significantly higher than that in other leaf types,and oxalic acid,trans Aconitine content was relatively high;the content of the sulphate and cis-aconitic acid in the Paeonia lactiflora leaf type was relatively high;the content of fucoidic acid,cis-aconitic acid and trans-aconitic acid in bamboo leaves was relatively high.There was no significant difference in the content of L-malic acid and succinic acid in different leaf types.(2)transcriptome sequencing of Pinellia ternata Transcript sequencing analysis of four leaf types of Pinellia ternata,Paeonia lactiflora leaf type,peach leaf type,bamboo leaf type and willow leaf type,was carried out by Illumina HISeq 2000 sequencing platform.A total of 53.22 Gb Clean Data was obtained by sequencing,and 126391 transcriptions were obtained by assembly.The total length was 105,613,896 bp,and the average read length was 835 bp.Through KEGG database comparison,there were 8 metabolic pathways with a total of 723 genes involved in alkaloid metabolism,and 31 metabolic pathways including 8709 genes involved in organic acid metabolism.(3)Differential expression analysis and gene mining of metabolic related functional genes in different leaf types of Pinellia ternata The expression of Pinellia ternata was significantly different in different leaf types.Taking the Paeonia lactiflora leaf type of Pinellia ternata as a control,the differentially expressed genes of Peach Leaf,Bamboo Leaf and Willow Leaf were annotated to 110,110 and 118 metabolic pathways respectively,and the number of differentially expressed genes was 1206,1341 and 1434,respectively,among which the number of differentially expressed genes enriched in metabolic pathways was 509,605,and 790.By qPCR,the relative expression levels of the 15 candidate genes were consistent with the transcriptome results,indicating that the transcriptional sequencing results were accurate and reliable.The content of organic acids in Pinellia ternata with Peach Leaf was the highest among different leaf types,which was closely related to the differential expression of pckA,MDH1,ACO,CS,ACLY and other enzyme genes.The discovery of related functional genes provides data support for the excavation and molecular-assisted breeding of new materials with high content of organic acid in Pinellia ternata.In summary,in this paper,the targeted research was carried out from the quality standard upgrading of Pinellia Rhizoma,grading standards of medicinal materials,grading standards and stem propagation of Pinellia ternate for seeding,and the correlation between inheritance and quality of different leaf types.It laid the foundation for comprehensively improving the quality of medicinal materials,excavating high-quality resources of Pinellia Rhizoma,and achieving the high quality with corresponding price in Pinellia Rhizoma.