| Siegesbeckia Herba(SH),a traditional Chinese medicine(TCM)used for the treatment of rheumatoid arthritis,is the dried aerial part of Siegesbeckia pubescens Makino,S.glabrescens Makino,and S.orientalis L.of the Asteraceae family.The sources of the SH are all wild,and the morphologies of these three species are extremely similar,making it difficult to accurately distinguish between them using traditional identification methods.Commercial SH is often a mixture of several different species.In recent years,there has been a significant increase in TCM preparations containing SH as a component,and the chemical components of different origin SH are also different,making it more urgent to conduct research on the pharmacologically active substances of the different origins.During the flowering period of SH,33 batches of samples were collected from various parts of the country.DNA barcoding was used to identify the origin of SH at the molecular level.Firstly,the extraction method for genomic DNA of SH was investigated.The total DNA obtained by the above extraction method was subjected to PCR amplification and bidirectional sequencing to obtain ITS1-5.8S-ITS2,ITS2 and pbs A-trn H sequences,respectively.The three sequences were compared by BLAST homology sequence analysis with the Gene Bank database.The results showed that among the 33 batches of SH samples collected,there were 10 batches of S.pubescens,10 batches of S.glabrescens,and 13 batches of S.orientalis.Based on this,Clustal W method was used to compare the gene sequences,and two phylogenetic trees of the three sequences were constructed based on standard parameters.The sequences were also analyzed for single nucleotide polymorphisms(SNPs),and the SNP analysis results showed that there were 18 SNP sites and 2 deletions between the three species from 21 bp to 685 bp,and there was no intra-specific variation in the sequences of S.glabrescens and S.pubescens,but there were three variable sites and two deletions in the sequence of S.orientalis.This further proves the feasibility of using DNA barcoding molecular identification technology to identify SH.Based on the results of DNA barcoding,SH were analyzed and identified for their chemical components using ultra-high-performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry(UPLC-ESI-QTOF-MS)and highperformance liquid chromatography-solid phase extraction-nuclear magnetic resonance(HPLC-SPE-NMR)techniques.Guided by UPLC-ESI-QTOF-MS,two known diterpenoids and a novel diterpenoid were extracted and isolated from S.glabrescens and S.pubescens with high purity,and their chemical structures were determined by various spectroscopic techniques to be 16-O-malonylkirenol(25),15-O-malonylkirenol(26),and 15,16-di-Omalonylkirenol(29),respectively.A total of 48 compounds were identified by comparing the mass spectrometry data with reference standards and spectra from databases and literature,including 8 phenolic acids,2 sesquiterpenoids,24 diterpenoids,8 flavonoids and 6 oxylipins.Using partial least squares discriminant analysis(PLS-DA)method,the accuracy of the origin identification results of DNA barcoding was further verified,and 12 chemical components were identified,including 4 characteristic compounds of S.orientalis,2characteristic compounds of S.glabrescens,4 common marker compounds of both S.glabrescens and S.pubescens,while 1 common marker compound of both S.orientalis and S.pubescens.A rapid thin-layer chromatography method for identifying the three species of SH was also established simultaneously.The effects of kirenol and three malonyl-substituted kirenol on LPS-induced RAW264.7macrophage migration were evaluated using the Transwell assay at non-cytotoxic concentrations.Compared to the blank group,RAW264.7 macrophage migration was significantly enhanced after LPS induction for 24 hours(P <0.01).Within the concentration range of 10-40 μM,both kirenol and the three malonyl-substituted kirenol significantly reduced LPS-induced RAW264.7 macrophage migration(P <0.001).Under the same dosing conditions,the ability of monosubstituted malonyl kirenol to inhibit cell migration was stronger than that of kirenol and bisubstituted malonyl kirenol.The inhibitory activities of JAK1 and JAK3 kinases were determined using a migration rate assay,and the results showed that monosubstituted malonyl kirenol could partially inhibit the kinase activities of JAK1/3.This study identified three species of SH at different levels using DNA barcode,UPLCESI-QTOF-MS,and TLC.The results of all three methods were consistent.Both UPLC-ESIQTOF-MS and TLC results showed that the 13 batches of S.orientalis did not contain kirenol,which did not meet the quality standards of SH in the 2020 edition of the Chinese Pharmacopoeia.Therefore,the rationality of using kirenol as a quality control component for S.orientalis deserves further discussion.This study provides a reference basis for the quality control of SH and the production of its preparations in TCM. |
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