Identification And Quality Of Epimedium Herbs Rating Category And Snakes

Goal:The Chinese Pharmacopoeia (ChP,2010version) includes Herba Epimedii that has various complex species, and snakes that has many counterfeits. It’s quite challenging to conduct comprehensive, effective, and objective quality control of them. Therefore, these two Chinese Medicines are chosen as representatives from plants and animals representatively for multi-dimentional analysis, to provide reference for authentication and quality control of Chinese Medicine made from plants and animals included in the ChP, as well as to provide supplement for the improvement of the standards used in ChP.Methodology:In this research,120samples were chosen from18species of Epimedii with5of them included in ChP; while79samples from20species of snakes were collected. The analysis of these samples included morphological characteristics authentication, molecular authentication, chemical authentication, and quality control, etc. The feasibility of applying DNA barcoding in the authentication of Herba Epimedii was further investigated with validation using BLAST, Kimura-2-parameter (K-2P) distances analysis, phylogenetic tree (NJ, MP, and ML), and "barcoding gap". The RRLC-DAD-ESI-MSn and UPLC-Q-TOF-HDMS methods were adopted to analyze the chemical composition of the samples from18species (5of them are included in ChP) of herba Epimedii. In combination with chemometrics analysis (similarity analysis, cluster analysis, and PCA for component analysis), the fingerprints of113samples of Herba Epimedii from18species were established and quantitative determination of five flavonoids (Epimedin A\B\C, Icariin, Baohuoside I) were used to evaluate the quality of Epimedium. In the meanwhile, QAMS method was adopted to measure the content of these five flavonoids (Epimedin A\B\C, Icariin, Baohuoside I). The quality of Epimedium included and excluded in the ChP was compared and evaluated. To avoid "false positive" that was found in the authentification of Zaocys using PCR which is recommended by ChP, the feasibility of applying mitochondrial DNA barcoding in the authentication of snakes was further investigated and validated using BLAST, Neighbor-Joining tree, K-2P distance, and "barcoding gap"Results:Five DNA sequences, ITS, psbA-trnH, rbcL, matK and rpoCl, were used for analysis and it was found that psbA-trnH had the highest rate of success. The DNA barcodes using ITS, psbA-trnH amd matK sequence could all have fully accurate identification of Epimedium koreanum. High Definition Mass Spectrometry (HDMS) was used to obtain the precise molecular mass of the compounds and the characteristics of fragment ions provided by MSn. These two pieces of information were then compared with standard substances or literature to identify23compounds including22flavonoids and one alkaloid. The chemical experiment tests showed that Epimedium pubescens and Epimedium brevicornu had the best quality; their contens in total five flavonoids were higher compared with Epimedium koreanum. Epimedium koreanum had stable quality, but its content in effective flavonoids was much lower compared with other Epimedium included in ChP, with qualification rate of5.88%only according to the standards of ChP. This meant the Epimedium koreanum had lower quality though it had stable species. Epimedium sagittatum has a broad distribution, but its quality is not stable, especially in Anhui province where it has a big production; the content of Icariin of Epimedium sagittatum in other locations was much lower. The source places of five wild Epimedium included in ChP were ranked based on the high-low order of similarity, and this could provide reference for their cultivation in selecting high-quality species. The cluster analysis based on both chemical componets and DNA barcoding using ITS, psbA-trnH, matK sequences showed that Epimedium koreanum could have complete cluster, and could be distinguished from other Epimedium. According to chemical experiment test and similarity analysis using fingerprints, Epimedium koreanum was stable in chemical components and their contents; Epimedium koreanum had higher similarity, while other Epimedium had large intra-specicies variations, theirfore, the inter-and intraspecies differences were not obvious. The QAMS method in the measurement of Epimedium included in ChP was investigated. It was found that different Epimedium included in ChP had large variations; Epimedium wushanense and Epimedium sagittatum were not suitable for contens measurement using QAMS method, due to their low contents in Icariin or lack of other flavonoids components. The platform using mitochondrial DNA barcoding based on16S, Cytb, and COI was established for snakes (Zaocys dhumnades, Bungarus multicinctus, and Agkistrodon acutus) included in ChP, and it is the first that16S is recommended as the best candidate sequence in quick assessment of snake medicine using DNA barcoding technique.Conclusion:This research had explored multi-dimensional analysis of Epimedium and snake medicine. The research findings could provide reference in the authentication and quality control mode for plant and snake medicine included in ChP, and could also provide experimental test reference in improving the standards used in ChP.