| Corydalis tomentella Franch.,a species of the genus Corydalis in Papaveraceae,has been used as a folk herbal medicine for the treatment of hepatitis and cirrhosis.However,its chemical composition is still unclear,and pharmacological effect lacks basic data,which seriously restricts the clinical application.Based on the methods and techniques of Herb genomics,phytochemistry and pharmacology,we studied the identification of medicinal materials,the active ingredients and main pharmacodynamics.This study provided the evidence for the clinical applications,it is of great significance for the artificial cultivation and the biosynthesis of important compounds.1.The largest sample size of Corydalis was used for identification of C.tomentella and its related species by DNA barcoding.Three regions?ITS,ITS2,matK?with high identification efficiency were screened out from five DNA barcode?ITS,ITS2,matK,rbcL and psbA-trnH?of nuclear genome and chloroplast genome.Based on 131 samples of Corydalis,different region and analysis methods were compared to screen the most suitable barcode and its analysis method.The result shows that,ITS?65.9%?provided the highest identification efficiency in single sequence,ITS+matK?69.6%?shows the highest identification efficiency in single sequence and its combination,and BLAST exhibited the highest identification efficiency in all analysis.DNA barcoding can effectively identify C.tomentella and its related species.2.High-throughput sequencing of the first chloroplast genome of Corydalis for identification of C.tomentella and its related species.C.tomentella and C.saxicola were sequenced by Illumina HiSeq4000 high throughput sequencing platform The 190 kb chloroplast genome of C.tomentella and 189 kb of C.saxicol were obtained.A total of 107 genes were annotated,including 73 protein-coding genes,25 tRNAs and 4 rRNA sequences.The chloroplast genome of C.tomentella exhibited the special structure,highly expanded IR region and extremely contracted SSC region?9636 bp?,as well as high proportion of scattered and tandem repeats.The MLtree was established by 70 common protein coding gene sequences.The result showed that C.tomentella was closely related to C.saxicola,but far from Papaver somniferum L.and Macleaya cordata?Willd.?R.Br.,and C.tomentella and C.saxicola can be identified succesfully by chloroplast genome.3.High-throughput sequencing of the nuclear genome of C.tomentella and high-quality genome been obtained.We performed whole genome sequencing for C.tomentella and generated 27.64 Gb??107×coverage?raw data,filtered subreads 4,077,968,N50 9.63 kb.The genome were estimated as a extremely low level of heterozygosity?about 0.3%?and 258.56 Mb of size.Finally,252Mb genome was assembled,covering 97.30%of predicted genome size.The C.tom entella genome showed 95.70%consistent assessments using BUSCO embryophyta subset.In total of 25,595 Protein-coding gene models were annotated.The 84.8%annotation completeness with 15.2%missing BUSCOs were assessed for C.tomentella protein sequences.CAFE analysis showed that there were 1374 expansion genes and 2532 contraction genes in C.tomentella.According to the standard clock of time tree and the evolutionary node of phylogenetic tree,the differentiation time of C.tomentella and M cordata was calculated as 36.48 million years ago.C.tomentella was cultured with 13C stable isotope labeled tyrosine.The result shows that 21 of the 22 compounds were identified by HPLC-Q/TOF MS in the downstream metabolic pathway,which confirmed the tyrosine pathway was the possible pathway of alkaloid biosynthesis of C tomentella.22 key enzymes related to alkaloid biosynthesis such as dehydrocavidine were screened from the genome of C.tomentella,the analysis of transcriptome and alkaloid content of different parts?roots,stems,leaves and flowers?showed that the expression of key enzymes genes was significantly correlated with the content of alkaloid.4,Isolation and identification of 6 alkaloids from total alkaloids in C tomentella for the first time.The monomer alkaloids were separated and prepared by preparative liquid chromatography.The compounds were identified by UPLC-ESI-Q-TOF-MS and AM600 NMR.6 monomers were isolated and their structures were identified as:dehydrocavidine,dehydro apoca vid ine,dehydro iso apocavidine,coptisine,dehydrocheilanthifoline and wore nine.The chemical constituents of C.tomentella from different parts were significantly different.Dehydrocavidine is the main active ingredient of C.tomentella,the content was higher in flowers and roots than the content in stems and leaves,whereas another ingredient,Coptisine,only distributed in flowers.There was no significant difference in chemical constituents between wild and cultivated C.tomentella,which indicated that cultivated C.tomentella could be used as a substitute for wild herbs.The chemical constituents of different harvesting stages were significant different,the highest content was found in March.5.We proved that C.tomentella has significant protective effect on alcoholic acute liver injury and strong anti-hepatitis B virus activity.The models of Alcohol-induced Acute Liver Injury and carbon tetrachloride-induced Acute Liver Injury in mice were used to evaluate the hepa to protective activity of aqueous extract of C.tomentella.The result shows that the aqueous extract could significantly reduce the ALT and AST activity in mice with acute alcoholic liver injury?P<0.01,P<0.05?at different doses?200 mg/kg,400 mg/kg,600 mg/kg?,and the highest activity was found at low doses?P<0.01?,which was better than that of bicyclol positive control.The result of carbon tetrachloride-induced Acute Liver Injury shows that aqueous exti*act of C.tomentella provided a weak inhibitory effect on ALT activity in acute liver injury induced by carbon tetrachloride at low dose?200 mg/kg?,but no inhibitory effect was observed at high dose.In this study,we performed detailed researches on a traditional C hinese medicine with definite curative effect.Our researches provided an effective method for identification of medicinal materials and revealed the substance basis of pharmacodynamic.Moreover,which clarified the main pharmacological activities,and provided scientific instruction for formulating local and national drug standards. |
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