Study On The Indentification Of Rhizoma Corydalis And Its Adulterant

ObjectiveUnder the guidance of traditional Chinese medicine (TCM) theory,the methods that indentification of Rhizoma Corydalis and its adulterant used modern analytical technique andmolecular biological technique was conducted. Building and improving the way of the thin layer chromatography (TLC) of Rhizoma Corydalis, building fingerprints by high performance liquid chromatography (HPLC) and fourier transform infrared (FTIR)。To compare the performance of the sequences which frequently-used to identify plant medicinal materials and confirm the DNA barcoding for Rhizoma Corydalis. To design of the specific primers on the based of the compare and use the allele-specific PCR to identify Rhizoma Corydalis and its adulterant with the specific primers.Methods1. TLC of Rhizoma Corydalis which existing was improved, increase the kinds of characteristic reference substance to be sure the method more suitable for identify Rhizoma Corydalis and its adulterant.2.IR spectroscopy were used to analyze and identify the fingerprints of Rhizoma Corydalis.the similarity of IR spectrum and cluster analysis of the 39 batches of samples were calculated.3. HPLC were used to analyze and identify the fingerprints of Rhizoma Corydalis and its adulterant. The similarity of HPLC of 39 batches of samples were contrast to identify them.4. To compare the performance of 4 sequences of Rhizoma Corydalis with PCR and confirm the DNA barcoding for it.5. To used allele-specific PCR to identify Rhizoma Corydalis and its adulterant with the specific primers which be designed base on the molecular biology study.Results1. Optimized the method of TLC. Enhanced the specificity to be easier to identify Rhizoma Corydalis and its adulterant by adding 2 kinds of specific standard substances.2. The IR fingerprint of Rhizoma Corydalis was established, depending on 26 batches of samples. To analyze the similarity and cluster analysis result that similarity of 26 batches of Rhizoma Corydalis were above 0.90,13 batches of adulterants were below 0.90.3. Optimized the method of HPLC, to shorten analysis time. The common pattern of HPLC fingerprints was established and 17 common peaks were marked with 26 batches of Rhizoma Corydalis samples. The similarity calculation resulte of the HPLC fingerprints of 39 batches of samples showed that similarity of 26 batches of Rhizoma Corydalis were above 0.90,13 batches of adulterants were below 0.85.4. Established the methods of PCR of 4 DNA sequences. Appraising the performance with genetic distance analysis, barcoding gap and tree-based method, it showed that ITS2 sequence was effectively used to identify Rhizoma Corydalis and its adulterant and this sequence was suitable for the DNA barcoding of Rhizoma Corydalis.S.Designed a pairs of primer by find out the different loci of ITS2 sequence. Identifing Rhizoma Corydalis and its adulterant by using allele-specific PCR with these pairs of primer.ConclusionBy studying the TLC, IR, HPLC fingerprints and DNA barcoding of Rhizoma Corydalis and its adulterant and established multiple kinds of identification method. This study provided a feasible standard for quality control, and also provided experimental basis for market supervision and safety of drug, and gave reference for the application development of Rhizoma Corydalis.