Identification Technology Of Lysimachia Christinae And Adulterants

Objective In view of the present situation of numerous Lysimachia christinae and adulterants and chaotic clinical use,the analysis methods of medicinal materials,powder microscopy,TLC,HPLC,UV and molecular identification.To identify Lysimachia christinae and Adulterants of Lysimachia hemsleyana,Centella asiatica,Glechoma longituba,Dichondra repens,Desmodium styracifolium.Affirm the main identification characteristics,and establish a method with strong specificity,wide application and high accuracy.Methods ①Through the methods of eye contact and hand touch,the character identification was carried out respectively to compare the similarities and differences in shape,size,color,smell and so on.②Powder microscopic observation was used to compare the similarities and differences in the types,structures,colors and other aspects of the contents of Lysimachia christinae and adulterants.③Use Quercetin and Kaempferin as the control substances,the TLC method was used to identify Lysimachia christinae and adulterants,and the differences in Quercetin,Kaempferin were compared according to their spots.④Use HPLC to study of Lysimachia christinae and adulterants,similarity comparison and cluster analysis were carried out to distinguish the positive and false fingerprints.Similarly,similarity comparison and cluster analysis were carried out on the samples from different producing areas to determine their authenticity.⑤With Rutin as the index,the total Flavone content of Lysimachia christinae and adulterants was determined by UV,so as to investigate the difference in the total flavone content and to study the antioxidant activity of ethanol extract of Lysimachia christinae in vitro.⑥Extract Lysimachia christinae and adulterants genome by PCR for amplification and sequencing,rbcL and ITS2 sequence to calculate its base composition,length,and analyze the information site,mutation site etc,based on calculated Lysimachia christinae K2P model and mixed falsify the genetic distance between,build the NJ tree of clustering analysis,is to distinguish the falsify.Results ①In character,the difference is mainly manifested in the aspects of stem and leaves,etc.,the leaves of Lysimachia christinae are opposite,heart-shaped or broadly ovate,the whole border,the main veins protrude obviously,after being immersed in water,the black and brown stripes can be seen through the light perspective.②In powder,the main differences are as:glandular scale,non-glandular hair,etc.,it contains red-brown glandular scale,thin-walled cells contain red-brown substances,occasionally non-glandular hair,and the non-glandular hair,stone cells,crystal,and pigment blocks of pseudo-mixed materials are more.③On TLC,the difference was as:there were two yellow-green fluorescent spots in Lysimachia christinae with the same position and same color,,but there were no spots in the same position and color except for Lysimachia hemsleyana.④The parameters of HPLC were optimized,and the HPLC fingerprint patterns of 77 batches of Lysimachia christinae and adulterants from different places were established.Identified 8 common peaks,and no.4 peak was Rutin and no.7 peak as Quercetin.The similarity degree was not high,the similarity degree of 59 batches adulterants of Lysimachia christinae was lower than 0.60,the similarity degree of Lysimachia hemsleyana was the highest,and the RSD of its common peak area was between 60.52%-129.02%.According to cluster analysis of 77 batches of Lysimachia christinae and adulterants can be classified into 5 categories,J1-J7 gathered for the first class,the class are Lysimachia christinae and Lysimachia hemsleyana,L1-L2 get together for the second,the class is Glechoma longituba,M1-M7 gather for the third class,the class is Dichondra repens,the X1-X7 gather for the fourth class,the class is Centella asiatica,G1-G7 as the fifth class,the class is Desmodium styracifolium.The similarity of 18 batches Lysimachia christinae from different place was between 0.837-0.955,and the RSD of its common peak area was between 2.78%and 20.24%.The similarity of from Sichuan was higher than 0.9,and that from Guizhou was 0.837.According to the result of cluster analysis,18 batches Lysimachia christinae samples can be divided into 2 categories,J1-J5 clustering is the first category,which is the sample from Sichuan province,and J6 is the second category,which is the sample from Guizhou province.⑤In the determination of total flavonoids content by UV,the extraction process was optimized,and the total flavonoids content of Lysimachia christinae was significantly higher than adulterants,which were as follows:Lysimachia christinae>Desmodium styracifolium>Lysimachia hemsleyana>Centella asiatica>Dichondra repens>Glechoma longituba.There was little difference in different batches from the same area,but there was significant difference in different areas,ranging from 1.084%to 2.621%.The content of total flavonoids in Sichuan was higher than other areas.The DPPH and hydroxyl were scavenging of the flavone of Lysimachia christinae,in a certain range,the scavenging capacity was dose-effect related to the concentration.⑥The length of ITS2 and rbcL sequences were 444 bp and 553 bp,the average GC content was 56.34%and 42.70%,and the intra species variation rate was 2.48%and 0.90%,respectively.There were 10 and 4 haplotype sequences,and there was an obvious interval between the ITS2 and its counterfeits.The NJ tree can clearly distinguish Lysimachia christinae and adulterants,and the 6 samples fromZhejiang province form a separate branch.Conclusion The 5 methods can be used to identify Lysimachia christinae and adulterants.Among them,the character,powder microscopic,TLC is the most convenient,has the simple,the quick characteristic,but has the separation effect poor,the automation degree low shortcomings;UV method for the determination of total flavonoids has the advantages of simple operation and good reproducibility,but the separation effect is not obvious.HPLC fingerprint method can reflect the specific chemical composition information and characteristic peak spectrum information,but it has some disadvantages such as complex extraction and analysis,time-consuming experiment,high reagent consumption,and high requirements for software equipment.Molecular identification technology is currently the most accurate method for species identification,which can achieve sensitive,convenient,rapid and accurate batch identification,but the corresponding software equipment has high requirements,more costs and other shortcomings.To sum up,a more appropriate analysis method can be selected according to different experimental conditions and their respective needs to achieve the purpose of effective identification.