| ObjectiveAiming at the phenomenon that there are numerous adulterants and confusion in clinical use of the traditional Chinese medicine Celosiae semen,a systematic study on the identification methods of Celosiae semen with small fruit seeds is carried out to supplement the existing identification methods.It is expected to provide new research ideas for the accurate identification and quality control of small fruits and seeds Chinese materia medica represented by Celosiae semen,and provide scientific basis and guarantee for seed identification and clinical medicine safety of Chinese medicine planting industry.Methods1.Through the identification of traits,micro traits,and microscopy,the authenticity identification of the medicinal materials and their contaminants of Celosiae semen and its adulterants were carried out,and their similarities and differences in appearance,type and tissue structure of inclusions were compared and analyzed.2.Based on Baidu’s customized artificial intelligence training platform EasyDL,using the micro-character pictures of Celosiae semen and its adulterants as training data to train the classification model of Celosiae semen and its adulterants.A method for quickly identifying based on artificial intelligence and machine vision technology is established,and import the application into the WeChat applet for promotion.3.Through DNA barcode analysis,compare the similarities and differences of the DNA information of Celosiae semen and its adulterants,establish and optimize the SCoT-PCR system to perform PCR amplification on Celosiae semen and its adulterants,and screen primers that can amplify specific segments of Celosiae semen,recover,clone and sequence the specific fragments,design primers based on the DNA information of the specific fragments and verify the specificity of the primers.4.IScreen for the specificity obtained from the SRAP marker of Celosiae semen primers were designed for the specific fragments.After establishing and optimizing the PCR reaction program and system,samples were collected,and DNA was extracted,specific primers were used to amplify and electrophoretic detection of DNA from Celosiae semen and its adulterants from different places,identification of authenticity and adulteration based on the size and presence of specific bands,and inspection and verification of its detection limit,applicability and accuracy.5.Using Agilent Chromatographic Column Eclipse Plus C18(250 mm×4.6 mm,5μm),flow rate:0.8 mL·min-1,column temperature:19℃,detection wavelength:201 nm,mobile phase:volume fraction of 0.1%phosphoric acid Solution(A)-Acetonitrile(B),perform gradient elution,identify Celosiae semen and its common counterfeit products based on HPLC fingerprinting technology,compare the counterfeit products with the genuine control maps,and compare different ratios(5%-20%)to identify the adulteration behavior,clarify the difference between the chemical composition of the Celosiae semen and its adulterants,so as to effectively distinguish it,and provide a reference for the quality evaluation of Celosiae semen and its adulterants.6.Use wet digestion to pre-treat the samples of Celosiae semen and its adulterants,use ICP-MS technology to determine the content of various inorganic elements in the samples,using SPSS 16.0 software for cluster analysis and SIMCA-P 14.1 software for principal component analysis,study the types,content and distribution of inorganic elements,and analyze the differences of inorganic elements.Establish a characteristic fingerprint of inorganic elements.Results1.There is little difference between the traits of Celosiae semen and its adulterants,and it is difficult to distinguish only by trait identification;the micro trait identification method magnifies the fine appearance traits of seeds,such as the shape,surface texture,and position of the seed,the difference is more obvious.The characteristics of Celosiae semen and its adulterants can be distinguished;the distinguishing characteristics of the longitudinal section of Celosiae semen and its adulterants are not obvious,but in the powder microscopic identification of the seed coat,the color and morphology of the outer epidermal cells provide an important basis for the microscopic identification of Celosiae semen and its adulterants.2.The in-depth learning model built by EasyDL,the image classification accuracy rate of Celosiae semen and its adulterants can reach about 93.7%-94.8%.For the medicinal material images collected by this system,the recognition rate of the WeChat applet has reached 80%-100%.The model can accurately identify the types of medicinal materials in the image under the same image collection environment of decoction pieces,and has a relatively stable and better recognition effect.3.Based on the results of cluster analysis of ITS2 sequence,Celosiae semen can be separated from C.cristata L.,A.tricolor L.,A.spnosus L.and A.retroflexus L.,but cannot distinguish between Celosiae semen and C.cristata L..ITS and trnL sequences can accurately distinguish five species of Celosiae semen,C.cristata L.,A.tricolor L.,A.spnosus L.and A.retroflexus L..The psbA-trnH,matK,rbcL sequence can only distinguish part of the samples.Therefore,by evaluating the effectiveness of ITS,psbA-trnH,rbcL,matK,and trnL in the identification of Celosiae semen and its adulterants,ITS sequence is recommended as DNA barcode candidate sequences of Celosiae semen and its adulterants,with trnL sequence as a supplementary sequence.4.From 36 SCoT primers,5 primers(S21,S22,S29,S30,S35)with specific fragments of Celosiae semen were screened out.Select the S21 primers to recover,clone and sequence their specific fragments.According to their sequence information,6 pairs of primers were designed,but the specificity of the primers disappeared;the specific fragments obtained from screening based on SRAP markers were used to design Specific PCR,and when the annealing temperature was 60℃ and the number of cycles was 30,a single bright band appeared at 206 bp and 581 bp in Celosiae semen and C.cristata L.respectively,and there was no band in other adulterants and blank controls.The minimum detection limit of this method for Celosiae semen is lng;the detection limit for Celosiae semen being adulterated with C.cristata L.is 2%.5.Using high-performance liquid chromatography fingerprinting technology to identify Celosiae semen,C.cristata L.,A.tricolor L.,A.spnosus L.and A.retroflexus L.,and found that there were 19 common peaks in 10 batches of Celosiae semen;the similarity between the HPLC fingerprints of the samples of Celosiae semen and the common control pattern is above 0.9,but the similarity between the samples of Celosiae semen is 0.852-0.992;The similarity of Celosiae semen and its adulterants is only 0.155-0.651,and the characteristic peaks of C.cristata L.,A.tricolor L.,A.spnosus L.and A.retroflexus L.did not appear in Celosiae semen at 44-48 min.Except for the 5%C cristata L.being doped Celosiae semen,the similarity is greater than 0.80,and the results of other adulteration ratios have obvious similarities(all are below 0.80).6.Using ICP-MS technology to determine the inorganic elements of Celosiae semen and its adulterants,using SPSS 16.0 software for cluster analysis and SIMCA-P 14.1 software for principal component analysis,both genuine and fake products can be analyzed.Use SIMCA-P 14.1 software to cooperate with ANOVA and rank sum test to identify different inorganic elements,and screen out 23 common inorganic elements,namely Li,B,Mg,Al,K,V,Cr,Mn,Fe,Co,Ni,Cu,Zn,Ga,As,Rb,Sr,Mo,Cd,Hg,Tl,Pb,Bi;At the same time,the inorganic element characteristic atlas of Celosiae semen and its adulterants containing 23 kinds of inorganic elements were established.The fingerprint and the content of Li,B,Mg,Al,V and other elements are quite different,indicating that the elements of Celosiae semen.C.crist at a L.,A.tricolor L.,A.spnosus L.and A.retroflexus L.are significant differences in content.Conclusion1.It is difficult to distinguish Celosiae semen and its adulterants only by appearance traits;the micro traits and powder microscopic characteristics of Celosiae semen and its adulterants are obviously different,which can be used as one of the important basis for their identification.2.Using artificial intelligence and machine vision technology,a deep learning-based classification method of Celosiae semen and its adulterants images was established.It enriches the research ideas of quality evaluation of traditional Chinese medicine,and provides experimental reference for the popularization of artificial intelligence in the field of traditional Chinese medicine identification.3.The ITS sequence is recommended as the DNA barcode candidate sequence of Celosiae semen and its adulterants,and the trnL sequence as the supplementary sequence;The SCoT primers designed for the specific fragments of Celosiae semen amplified by primer S21 have not been successfully developed and need to be further study;a specific PCR identification method based on SRAP marker development has been established,and the detection method has good accuracy and applicability.A PCR reaction can be used to identify Celosiae semen and C cristata L.,and whether Celosiae semen is adulterated with C.cristata L..The method is highly sensitive,and the detection limit for adulteration of C.cristata L.is 2%.It has the advantages of simple operation,fast speed and high specificity.4.There are certain differences in HPLC fingerprints of Celosiae semen from different producing areas,suggesting that the source of production should be fixed to ensure the stability of the quality of Celosiae semen;the fingerprints of Celosiae semen and its adulterants are significantly different,and the chemical composition information is different.It is not suitable to mix Celosiae semen with C.cristata L.,A.tricolor L.,A.spnosus L.and A.retroflexus L..5.There is a big difference between the inorganic components of Celosiae semen and its adulterants.The characteristic map of inorganic elements can be used to distinguish Celosiae semen and its adulterants.Some of the components are related to the curative effect of Celosiae semen,so it is not appropriate to mix them. |
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