DNA Barcoding Identification Of Traditional Chinese Medicine Snake Gallbladder And Study On The Anti-psoriasis Chemical Constituents Of Euphorbia Helioscopia

Part one Study on the DNA Barcoding Identification of Traditional Chinese Medicine Snake GallbladderSnake gallbladder（Shedan）,a precious traditional Chinese medicine material,possesses therapeutic effects of clearing away heat and detoxification,reducing phlegm,and relieving spasm.A variety of preparations containing snake bile are recorded in the Pharmacopoeia of the People’s Republic of China（Part one）（2020 edition）,but the quality standard of snake gallbladder crude drugs has not yet been established.To ensure the safety and effectiveness of the clinical application of snake bile,based on the qualitative and quantitative analysis,separation and identification of snake bile components from the work of the previous laboratory,the molecular identification of snake gallbladder was carried out in this study.Firstly,the gallbladder samples of commonly seen snake species were collected in batches from the producing areas of traditional Chinese medicine snake gallbladder,and DNA was extracted from their gallbladder tissue or bile.The COI（mitochondrial cytochrome c oxidase subunit I,COI）sequence was sequenced and further compared to the reference sequences in the Barcode of Life Data System（BOLD）and Gen Bank for sequence similarities,genetic distance and phylogenetic tree analysis were performed to establish a DNA barcoding identification method for snake gallbladder.Then,many batches of commercial crude drugs and the common counterfeit products of snake gallbladder were collected and identified.Meanwhile,in this study,a pair of mini-barcode primers were designed to amplify the DNA and obtain a short fragment of COI gene,and the DNA mini-barcoding technique was used as an auxiliary identification method.A COI sequence with a length of 658 bp（COI barcode sequence）was finally obtained by PCR amplification with SNCOI2 primers from each snake gallbladder sample and snake bile sample.In BOLD and Gen Bank,each sequence obtained was assigned to the same species,and the highest sequence similarity was not less than 98%.The results of genetic distance analysis of snake species displayed that the average genetic distance within species（0～0.0360）was much smaller than the average genetic distance between species（0.1716～0.2272）,and the maximum intra-species genetic distance（0.0814）was also smaller than the minimum inter-species genetic distance（0.0908）.There was no overlap between intra-and inter-species genetic distances,demonstrating the obvious barcoding gap in snake species.Phylogenetic tree analysis yielded Bayesian Inference tree and neighbor-joining tree with similar topological structures,and the snake species of each family were clustered separately in both trees,and the molecular identification results were consistent with the morphological classification of their original animals.The original species of commercial snake gallbladders samples were identified as 13 snake species belonging to four families（n=156）and chickens（n=9）;except the duck gallbladders,the other common counterfeit products from chicken,pig,cow and sheep had been accurately identified.With the mini-barcode primers SNCOIM,the DNA of each snake bile sample could be amplified,yielding the short fragments of COI gene with a length of 202 bp,the identification results of which were consistent with the COI barcode sequence.In all,89.74%（175/195）of the commercial samples of snake gallbladder were effectively identified through DNA barcoding and DNA mini-barcoding.However,the DNA extracted from all the counterfeit bile could not be amplified with the mini-barcode primers.The DNA barcode molecular identification system of snake gallbladder has been established in this study,in which the DNA mini-barcoding method contributes to authenticating the snake gallbladder crude drugs with degraded DNA.The original source of commercial snake gallbladder crude drugs has been preliminarily clarified,including the gallbladders of Xenopeltis unicolor belonging to the family Xenopeltidae other than Colubridae,Elapidae and Viperidae,possessing great significance for the original identification and quality control of snake gallbladder crude drugs.Part two Study on the Anti-psoriasis Chemical Constituents of Euphorbia helioscopiaEuphorbia helioscopia L.,a herb that belongs to the family Euphorbiaceae,the whole plant of which is used as a Chinese herbal medicine with the effects of diuresis and swelling-dispersing,expectorant cough,detoxification and insecticide.According to the previous phytochemical and pharmacological studies,diterpenoids of various structures may be the major bioactive components of E.helioscopia,among which the jatrophane-type diterpenoids were preferentially selected here for study to provide an anti-psoriasis material foundation of the herbal medicine.The diterpenoid components of E.helioscopia were separated and identified by conventional chromatographic techniques,high resolution mass spectrometry,1D and 2D NMR spectroscopy,ECD calculations,and X-ray single crystal diffractions.Meanwhile,the inhibitory effect of some isolated compounds against Kv1.3 ion channel was evaluated in a human embryonic kidney 293 cell model transfected with plasmid encoding Kv1.3.A total of 43 jatrophane diterpenoids and its analogues were isolated and identified,including 17 new jatrophane diterpenoids,namely,heliojatrone D（1）and helioscopids A-P（2-17）,along with 26 known analogues（18-43）.Structurally,heliojatone D（1）is the fourth natural diterpenoid with a rare bicyclo[8.3.0]tridecane skeleton.The results of in vitro activity screening showed that all the tested compounds had a certain inhibitory effect against Kv1.3 ion channel,among which compounds 2 and 15 demonstrated the most significant inhibitory activity with the IC₅₀ values of both 0.9μM.In this study,A series of components with Kv1.3 ion channel inhibitory activity has been found in E.helioscopia,revealing the traditional anti-psoriasis material foundation of the herbal medicine.