Identification Of The Origin Of Evodia Rutaecarpa And Study Of Its Medicinal Quality Based On Chloroplast Genom

Objective: To develop an accurate and effective method for th e identification of medicinal plants of the genus Euodia and to evaluate the quality of Euodiae fructus of different sources and specifications,providing a basis for further development and utilization of Euodiae fructus.Methods: 1.Literature search and fieldwork were used to investigate the original plants of Euodiae fructus.Fruit development of Euodia rutaecarpa,Euodia rutaecarpa var.bodinieri,and Euodia Rutaecarpa var.officialis was observed by somatic microscopy.2.Whole genome sequencing,assembl e and annotation of seven species of medicinal plants of the genus Euodia were performed using second generation high throughput sequencing technology.The chloroplast genome features were analyzed using MEGA,condon W,REPuter,and m VISTA software,and the ML phylogenetic tree of Rutaceae was constructed based on chloroplast whole genome sequences and shared protein coding genes sequences.3.The fingerprint profiles of E.rutaecarpa,E.rutaecarpa var.bodinieri,and E.Rutaecarpa var.officialis were established based on UHPLC method,and the chemical composition of Euodiae fructus was identified by mass spectrometry based on literature review combined with the comparison of reference substance(Neochlorogenic acid,Cryptochlorogenic acid,Chlorogenic acid,Dehydrocorydaline,Evodiamine,Rutecarpine,rutin,Hyperoside,Narcissoside)to identify the chemical constituents of Euodiae fructus,chemical pattern recognition and cluster analysis by SIMCA14.1 and IBM SPSS 26.0 software;the content s of Limonin,Evodiamine and Rutecarpine were determined by HPLC,comprehensively the quality of Euodiae fructus was evaluated.Results: 1.The genus Euodia is widely distributed in the south of the Yangtze River in China,and its main producing areas and varieties of herbs do not vary much,except for the species specified in the Chinese Pharmacopoeia,the fruits of the same genus were often used as substitute and adulterants of Euodiae fructus.But fruit morphological observ ations indicate a large variation in fruit morphology among species of Euodia.2.The chloroplast genomes of the seven medicinal plants of the genus Euodia measured were typical quadripartite structures with lengths ranging from 15856 3 to158762 bp.The length of LSC region was 86104-86273 bp,the length of SSC region was 18212-18265 bp,and the length of IR region was 27110-27127 bp.All chloroplast genomes were annotated to 133 genes,including 88 protein-coding genes,37 t RNA genes and 8 r RNA genes,of which 18 genes were duplicated in the IR region.The GC content was 38.33-38.37%.3.Through comparative genomic analysis,14 highly variable regions were identified,namely t rn H-psb A,psb Z-trn G,pet N-psb M,trn R-atp A,trn D-trn Y,psb C-trn S,trn S-psb Z,ycf4-cem A,rpl32-trn L,atp A,atp F,trn Y,rbc L and clp P.These sequence fragments can be used as potential DNA barcodes for the identification o f Euodia species.The phylogenetic relationship analysis results showed that two phylogenetic trees constructed by the complete chloroplast genome sequence and the shared protein coding gene data set were basically the same,and the former had higher resolution than the latter.The relationshi p between E.rutaecarpa and E.rutaecarpd f.meionocarpa was closer,and they gather into one branch;E.rutaecarpa var.bodinieri,E.rutaecarpa var.officinalis and E.compacta were sister species of each other.Them were more closely related and gather into one branch;These two main branches were sister groups of each other with 100% support.The genetic relationship between E.glabrifolia and E.fargesii was relatively close to one branch,and relatively far from the first two branches.4.A UHPLC fingerprinting method was developed to detect and obtain fitted control fingerprints for the three original plants samples(39 batches in total)of Euodiae fructus as specified in the Chinese Pharmacopoeia.11,13 and 17 peaks were shared in the fingerprints of E.rutaecarpa var.officialis,E.rutaecarpa var.bodinieri,and E.rutaecarpa,respectively,and the number o f peaks in the 39 batches of fitte d control fingerprints was 8.Total of 22 compounds were identified by the fingerprint profile peak designation,and 8 components(Neochlorogenic acid,Cryptochlorogenic acid,Chlorogenic acid,Dehydrocorydaline,Evodiamine,Rutecarpine,rutin,Hyperoside,Narcissoside)were identified by the reference substance.Chemical pattern recognition could classify 39 batches of Euodiae fructus into 3 categories,E.rutaecarpa,E.rutaecarpa var.bodinieri,and E.Rutaecarpa var.officialis into 1 category each;the results of cluster analysis were consistent with the results of chemical pattern recognition analysis;the cumulative variance contributions of the 2 principal component factors were 74.10% and91.80%,respectively,by principal component analysis.The re sults of content determination showed that among the 47 batches of herbs,the quality fraction of Limonin of Xiaohua specimen of Euodiae fructus ranged from 0.08% to 1.96%;the quality fraction of Evodiamine ranged from 0.04% to 0.28%;the quality fraction of secondary bases of Rutecarpine ranged from 0.06% to 0.21%;the quality fraction of Limonin of Zhonghua specimen of Euodiae fructus ranged from 3.53% to 3.68%;the quality fraction of Evodiamine ranged from 1.79%～2.51%;Rutecarpine mass fraction was 0.58%～0.72%;the content of the three components of the mixed forgeries was generally lower than that of the genuine Euodiae fructus.Conclusion: The chloroplast whole genome sequence can be used as a super barcode for the accurate identification of medicinal plants of the genus Euodia,and the screened chloroplast genome highly variable can be used as a specific barcode for the molecular identification of Euodia.The results of this study support the combination of E.rutaecarpa,E.rutaecarpa var.bodinieri,E.rutaecarpa var.officinalis,E.compacta and E.rutaecarpd f.meionocarpa into one species in Flora of China,but do not support the combination of E.glabrifolia and E.fargesii into one species.Fingerprinting combined with chemical pattern recogniti on can be used to distinguish different specifications of Euodiae fructus and to evaluate th e quality comprehensively.