Study On The Correlation Between Traditional “Assessing Quality By Distinguishing TCMM Features” And Modern Quality Evaluation Methods Of Polygala Tenuifolia

Rationales: Chinese medicinal materials as a special commodity,is an important raw material of Chinese medicine Pieces and Chinese patent medicine.During the long-term market transaction,the traditional experience that depend on "Rating level according to the quality of goods,setting price on the basis of different grades" to determine herbs quality was formed.Afterwards gradually formed a medicinal herbs commodity specifications after induction and summary,and took the commodity specifications as the standard to distinguish the quality of herbs.However,with the introduction and domestication of wild medicinal herbs and large-scale cultivation,the quality of Chinese medicinal materials,especially rhizome medicinal materials,has undergone great changes under the artificial over-intervention.Then,these drugs will be mistakenly understood in market circulation specification and quality identification,resulting in the unreasonable phenomenon of "the same price(quality)but different quality(price)","high price(quality)but no quality(price)",which can not ensure the consistency of clinical efficacy of Chinese herbal medicines,and seriously influence the long-term stability and prosperity of the pharmaceutical market.This study took Polygala tenuifolia Willd.as an example,which is one of Shanxi geoherbism drugs,with the traditional identification experience of herbal medicines as the starting point,that is,"assessing the quality by distinguishing the features of traditional Chinese medicinal materials".Combined with market and origin research,firstly revise the commodity specification standards of P.tenuifolia including the version released in 1984 of "seventy-six kinds of herbs commodity specifications".Then test and verify the rationality of commodity specifications grades for the evaluation of P.tenuifolia medicinal herbs by chemical analysis and DNA molecular marker,and put forward the scientific basis for the formulation of commodity standard of P.tenuifolia.Objective: 1.Revise the commodity specification standards of P.tenuifolia for its better circulation in the market.2.The quantitative analysis method of UPLC that "multi-component quantitative and fingerprint" was used to clarify the chemical difference between P.tenuifolia herbs with different specifications.And evaluated the rationality that assess the quality of P.tenuifolia herbs by the method of chemical analysis.3.Two DNA molecular marker techniques of ISSR and AFLP were used to construct the DNA fingerprint of P.tenuifolia,the aim of this study was to elucidate the relationship between the grade of P.tenuifolia,the chemical composition of P.tenuifolia and molecular bands of ISSR or AFLP.These provided a theoretical basis for the classification of commodity specifications of P.tenuifolia at the level of DNA.4.A specific ISSR sequence,which associated with the specifications and chemical composition of P.tenuifolia,was designed to a specific PCR primer that can rapidly identify the content of P.tenuifolia compounds,and then used for routine identification of high quality P.tenuifolia.Methods: 1.To conduct the market and origin research of the P.tenuifolia,and to revise the current commodity specification grade of P.tenuifolia.2.Using UPLC to determine the content of 6 sucrose esters and 4 xanthone,and establish the UPLC fingerprint of P.tenuifolia.Then the common peaks of UPLC fingerprint were assigned by means of integrated UHPLC-Q Exactive quadrupole-Orbitrap hybrid mass spectrometer.From the level of chemical analysis,the chemical differences between different specifications(wild and cultivated)and different grades(large tube and middle tube)were analyzed,and the rationality of the product specification grade of P.tenuifolia was evaluated by chemical means.3.The ISSR and AFLP molecular markers were used to construct the DNA fingerprints of P.tenuifolia,and the specific ISSR and AFLP molecular bands related to the product grade and chemical composition of P.tenuifolia were screened by correlation analysis.Finally,found the relationship between the commodity specification grade,chemical composition and the DNA molecules bands.4.The specific ISSR bands that related to the content of chemical composition and commodity specification grade were collected,cloned and sequenced,and the target gene sequence was analyzed.The specific PCR primers that were designed according to the target gene sequence were designed to rapidly identify the content of P.tenuifolia compound.Results: 1.According to the actual situation of the market and the origin research to revise the commodity specifications grade of P.tenuifolia,and put forward a commodity specification standard draft of P.tenuifolia medicine,which was more close to the actual situation and according with the market circulation.2.In this study,UPLC was used to determine the contents of six sucrose esters and four xanthone in P.tenuifolia,and the method is enough stable and reliable to evaluate the quality of P.tenuifolia.Comprehensive quality evaluation function of P.tenuifolia showed that the comprehensive quality of big tube herbs was better than that of medium tube herbs.Except for individual samples,the overall quality of wild P.tenuifolia was better than that of cultivated products.The results of the clustering of 10 compounds were the same as those of PCA,but the specifications and grades of the samples were not identical in the same cluster.The similarity of the UPLC fingerprints showed that the similarity of the samples was relatively small between different grades.These results illustrated that the commodity specification level could reflect the quality of P.tenuifolia,but only based on the product specifications rating the quality of P.tenuifolia medicinal herbs is more one-sided.3.Two DNA molecular marker technologies of ISSR and AFLP were used to establish the DNA fingerprints of P.tenuifolia.Using the correlation analysis to filter two ISSR bands significantly correlated with the specifications of P.tenuifolia,eleven AFLP bands significantly correlated with the grade of P.tenuifolia,thirty-one ISSR bands with significant correlation with the content of compound in P.tenuifolia,forty-one AFLP bands with extremely significant correlation with the content of compound in P.tenuifolia.The content of compound in P.tenuifolia can be speculated according to the specific DNA molecular bands.4.The ISSR bands,which were significantly correlated with the content,were executed by glue recovery,clone sequencing,and sequence function analysis,and two specific PCR identification primers were designed.The content of sibiricose A5,sibiricose A6,sibiricoxanthone B,glomeratose A,7-O-methyl mangiferin,polygalaxanthone III,tenuifoliside C can be calculated to identify the high quality of P.tenuifolia.Conclusion: This paper revises the current commodity specification of P.tenuifolia based on the results of market and origin research,and expounds the rationality of the commodity standard of P.tenuifolia used to assess the quality standard of P.tenuifolia through chemical analysis and DNA molecular marker technology.These results prove that the commodity standard can reflect the quality of P.tenuifolia.But it is difficult to get more comprehensive results only by the commodity specifications to evaluate the quality of P.tenuifolia.The two pairs of specific PCR primers designed in this study could predict the content of compounds in P.tenuifolia and could be used for routine identification of high quality P.tenuifolia.