Multidimensional Identification Methods And Equality Evaluation Of Trachelospermum Jasminoides

The authenticity identification and quality evaluation of Chinese herbal medicines are important guarantees for the safety and treatment effects of Chinese medicines.It's our new goals to develop more accurate,effective,convenient and practical methods of Chinese medicine identification and quality evaluation,by way of introducing and applying new technologies and methods to achieve these goals.In this paper,we took 101 copies of Trachelospermum jasminoides and its commonly used adulterants as research subjects and used DNA barcoding technology to identify the base accurately.Based on the study of DNA barcoding,we made a sensitive and rapid high-resolution melting curve identification,which was an effective molecular complement to DNA barcoding technology.Given that rapid,non-destructive testing is required in the market,we used Infrared Spectroscopy to make a tri-step identification for Trachelospermum jasminoides and its adulterants and HPLC fingerprints were used for further identification and quality assessment of medicinal materials.The main findings were as follows:1.101 high-quality ITS2 sequences of Trachelospermicum jasminoides and its adulterants were obtained.The success rate of amplification and sequencing were both 100%;the sequence length of Trachelospermicum jasminoides and its adulterants was 220 bp to 241 bp;the GC content was 61.3%-74.1%;the alignment length of the sequence was 267 bp,among which the number of mutation sites was 126.The minimum genetic distance between species was 0.488,which was much greater than the intraspecific genetic distance of 0.000.And there was an obvious barcoding gap between the authentic and adulterants.There were a central ring and four similar helixes ?,?,?,? in the ITS2 secondary structure of Trachelospermum jasminoides and its adulterants,but it made obvious differences in the number of stem rings,sizes,positions and the central angles of spiral arms.Furthermore,a phylogenetic tree was constructed.The ITS2 sequences of Trachelospermum jasminoides and its adulterants were clustered into one tree respectively,meantime LS006 and LS041 were found as two adulterants.At the species level,the identification success rate of ITS2 sequences against Trachelospermum jasminoides and its adulterants was 100%.Moreover,the two-dimensional DNA barcoding of Trachelospermum jasminoides and its adulterants was generated,which realized information transformation of sequences and made the application of DNA barcoding more simple and convenient.2.A high-resolution melting curve model for Trachelospermum jasminoides and its adulterants was established.There were three peaks in the melting curve of Trachelospermum jasminoides and Ficus pimtila,with the main peak at 92.34? and 86.95? respectively;there were two peaks in the melting curve of Ficus tikoua and Euonymus fortunei,with the main peak at 92.84? and 91.56? respectively.A stable melting curve could be obtained within the concentration range of 5-200 ng/?L for the DNA templates of Trachelospermum jasminoides and its adulterants,but the HRM obtained by the melting temperature peak method had a relative change in the peak position.The melting temperature peak method,the standard melting curve and its derivative curve can all accurately identify Trachelospermum jasminoides and its adulterants.The melting curve of Trachelospermum jasminoides sample LS006 was consistent with that of Euonymus fortunei;the melting curve of sample LS041 was consistent with that of Ficus pumila.These results above verified the identification results of DNA barcoding and we could conclude that the HRM technology can detect I%of the adulterants in Trachelospermum jasminoides.3.In the one-dimensional spectroscopy,there were C=O absorption peaks in the wave band 1647-1623 cm-1 of Trachelospermum jasminoides and its adulterants,but the peak positions were different,indicating different types of flavonoids.Trachelospermum jasminoides and its adulterants all had C=C skeleton vibration absorption peaks of aromatic rings near 1377 cm-1,a characteristic absorption peak of calcium oxalate near 13 18 cm-1,and a strong absorption peak of cellulose near 1034 cm-1.In the 1000-1250 cm-1 wave band,in addition to the common peaks,the position and intensity of the peaks of Trachelospermum jasminoides and its adulterants were different,indicating different kinds of flavonoids.In the second Derivative Infrared Spectroscopy,we could see the absorption peak of Trachelospermum jasminoides at 1736 cm-1.There were C=C skeleton vibration absorption peaks of aromatic rings near 1517 cm-1,a strong characteristic absorption peak at 1468 cm-1,a polysaccharide area at 855-1250 cm-',and a strong common peak at 1077 cm-'.In addition to the common peaks,the position and intensity of the peaks of Trachelospermum jasminoides and its adulterants were different,further indicating different types of glycoside compounds.In the tri-step identification,thermal perturbation had a great influence on the flavonoids and lignan compounds in the Trachelospermum jasminoides;the flavonoids and glycoside compounds in the Ficus pumila were greatly affected by thermal perturbation;contents in Ficus tikoua were generally little affected by thermal perturbation;relatively speaking,thermal perturbation had a greater effect on the ketones and lipid components of Ficus tikoua;and the flavonoids,alcohols and glycosides in Euonvaus fortunei were greatly affected by thermal perturbation.There were differences in the number of automatic peaks and the location of the strongest automatic peak in the wave band 1805-855 cm-1 of Trachelospermum jasminoides and its adulterants.The results of cluster analysis showed that Trachelospermum jasminoides and its adulterants were clustered into one tree respectively.At the same time,LS006 and LS041 were found to be adulterants,Euonymus fortunei and Ficus pumila respectively.This finding further validated the identification results of DNA barcoding.4.It was determined that(10:90)-(30:70)15 min-(40:60)40 min-(60:40)75 min was the best chromatographic condition.By comparing HPLC fingerprints of Trachelospermum jasminoides and its adulterants,it was identified that LS006 and LS041 were Trachelospermum jasminoides adulterants circulating in the market.Moreover,we established the control fingerprints of Trachelospermum jasminoides and performed the similarity assessment based on the common peak pattern.The results showed that there were 64 batches of Trachelospermum jasminoides with similarity above 0.90,accounting for 80%of the total samples;there were 9 samples with similarity between 0.85 and 0.90;and there were 7 samples with similarity less than 0.85.Then the content of colostrin in Trachelospermum jasminoides was determined.Based on the 2015 edition of the Chinese Pharmacopoeia Standard,there were 6 samples whose tracheloside content was less than 0.40%.Though they were all genuine products,they were unqualified.