Multi-dimensional Quality Evaluation Of Pogostemon Cablin From Different Varieties

ObjectiveThe quality of traditional Chinese medicine(TCM)is the basic guarantee of the clinical efficacy.However,it is difficult to achieve a comprehensive and reasonable evaluation of the quality of TCM by using certain method alone,such as character identification,molecular identification.The ideal quality evaluation method of TCM is supposed to combine characteristics,chemical composition and molecular biological evaluation to achieve multi-dimensional quality evaluation.Pogostemon cablin is used in dry upper parts which has the functions of eliminating turbidity,relieving vomiting and relieving heat.It is one of the famous "Top Ten South Medicines" and widely used in clinical practice.It is also an important traditional Chinese medicine in the prevention of CO VID-19(Corona Virus Disease 2019).P.cablin produced in different places have certain differences in characteristics,chemical composition,gene and the quality varies.Which cuases the complexity of the classification.In recent years,higher economic benefits have prompted the emergence of counterfeit species,causing irreparable economic losses and health hazards to consumers.Therefore,it is of great significance to evaluate the quality of P.cablin of different cultivars and chemical types accurately in the context of the current development of Southern Medicine Resources.The essential oil component of P.cablin is an important indicator of its quality,and glandular hair is the main place of the synthesis and secretion of essential oil.Studies have shown that the morphological characteristics of plant glandular hairs are a reliable basis for plant classification and their growth and development status can even affect the overall quality of TCM.This study aims to explore and establish a multi-dimensional quality evaluation system of traditional Chinese medicinal materials based on the combination of "characteristic evaluation+chemical evaluation+molecular evaluation".Inferior,provide a new method for the quality control of traditional Chinese medicine.At the same time,develop a simple and efficient method to distinguish P.cablin and its counterfeit species,to protect consumer rights and safety of clinical medication.Methods1.Character and micro shape identification and evaluation:Based on the epidermal hair characteristics of P.cablin leaves from 8 different varieties sources and 2 harvesting periods(end of June and end of October)were used as materials and chloral hydrate permeating method was used for observation.Microsoft Excel 2019 statistical data and SPSS 23.0 were used for variance analysis,principal component analysis and cluster analysis.2.Identification and evaluation of chemical composition:the content of patchouli and pogostone in the methanol-extracted part of P.cablin from different varieties was determined by GC and the average content was clustered by SPSS 23.0.Correlate the results with epidermal hair density to explore the relationship between characteristics and chemical composition.3.Molecular identification:DNA barcode technology was used to amplify and analyze ITS2 and matK sequences of samples from different varieties.SPAdes,GeSeq,mVISTA were used to assemble,annotate,and analyze the chloroplast genomes of P.cablin from different varieties based on second-generation sequencing technology.4.Identification of counterfeit species:Primer 3.0 software was used to design specific identification primers based on the ITS2 sequence,specific PCR reaction conditions was optimized.Results1.P.cablin from 8 different varieties and 2 harvest periods are divided into 4 categories,based on the characteristics of patchouli leaf epidermal fur(leaf epiderma 1 fur density and diameter),which is in good agreement with traditional classificatio n results.2.P.cablin from 8 different varieties and 2 harvest periods were divided into 3 chemical types based on the content of patchouli alcohol and patchoulenone which is consistent with previous research results3.DNA barcode sequencing analysis showed that samples from 8 different varieties were all P.cablin.The whole chloroplast genome of P.cablin from 8 different varieties was obtained;3 hypervariable regions were selected(rbcL,ndhB,rrn23).4.The identification primers designed based on the ITS2 sequence is of good specificity.The specific bands of genuine original plant,herbal medicine and Chinese patent medicine are clear and bright,and there is no banding for the counterfeits under optimized PCR reaction conditions.Conclusion1.A new classification method was established based on the leaf epidermal hair characteristics and the role of epidermal hair characteristics in classification of P.cablin n was confirmed.The application of epidermal hair traits in the classification of P.cablin varieties was supplemented.2.A new method for the determination of chemical type of P.cablin by GC was established which greatly improved the effectiveness of quality evaluation.The correlation analysis between the content of chemical components and the characteristics of epidermis hair showed that there was a certain correlation between them.The effect of glandular hair characteristics on the quality of P.cablin was verified,the ground formation mechanism was further revealed.3.The accuracy of the sources was verified;Compared with the research results of many years ago,P.cablin from different places and varieties has changed in genes after years of cultivation.The 3 highly variable regions of P.cablin chloroplast genome(rbcL,ndhB,rrn23)can be used as candidate DNA barcodes for identification of the cultivars and chemical types4.A specific PCR identification method was established,which can accurately identify P.cablin and its counterfeit species.This method is also applicable to Chinese patent medicines.In this study,a multi-dimensional quality evaluation system of traditional Chinese medicine materials was established by observing and analyzing the characteristics of leaf epidermis,determining the chemical content and molecular identification.The whole chloroplasts genome of P.cablin from 8 different varieties were obtained,which provided the basis for the research of super DNA barcode.The correlation between the characteristics of P.cablin epidermal hair and the content of chemical components was verified,and the mechanism of quality formation of P.cablin was further revealed.It provides a more comprehensive method for variety classification and quality evaluation of P.cablin,a explanation of the "distinguishing the appearance and discussing the quality" theory from the perspective of modern science and new ideas and references for methods of quality evaluation of traditional Chinese medicine.