ABSTRACT
Objective:To explore the effect of chemical compound of aconitum alkaloid on the lipopolysaccharide （LPS）-induced inflammatory response of RAW264.7 macrophages and investigate its mechanism. Method:The chemical compounds of Aconitum Kusnezoffii Reichb were collected from TCMSP database with consideration of oral bioavailability （OB）≥30% and drug-likeness （DL）≥0.18. The potential targets of each chemical component were predicted with use of Pubchem database and Swiss Target Prediction database. Rheumatoid arthritis （RA） targets were collected from GeneCards database and selected by intersection screening. Gene ontology （GO） classification enrichment and Pathway enrichment analysis were carried out with use of DAVID database. Cytoscape was used to construct "Chemical Compound-Potential Targets-Pathway-Disease" network. Protein-protein interaction （PPI） network was constructed by using STRING database and Cytoscape software. RAW264.7 macrophages were stimulated by LPS to establish macrophage inflammation model <italic>in vitro</italic>. Western blot was used to detect the effects of chemical compounds on the expression of tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>） and cyclooxygenase-2 （COX-2） in RAW264.7 cells induced by LPS， as well as on the expression of JAK kinase and nuclear transcription factor- kappa B （NF-<italic>κ</italic>B） signal pathway. Result:A total of 27 chemical compounds were obtained by searching TCMSP database and consulting literature （OB≥30%， DL≥0.18）. 12 chemical compounds were obtained after screening. 177 potential targets were obtained after database prediction and screening， and 97 targets were obtained as potential targets for the treatment of RA after intersection between 177 potential targets and 4 329 RA targets. A total of 32 biological processes （BP）， 5 cellular components （CC）， and 12 molecular functions （MF） were enriched by DAVID database. The construction of network topology map showed that different chemical compounds can act on the same target and the same chemical compound can also act on different targets in the treatment of RA. Aconitum alkaloid can be connected with the same pathway through different targets or with different pathways through the same target， indicating that different targets may have synergistic effect， which fully reflected the complex multi-compound， multi-targets and multi-pathways mechanism. Different concentrations of LPS in stimulation （0-200 μg·L<sup>-1</sup>） can significantly up-regulate the expression of COX-2 protein in RAW264.7 macrophages （<italic>P</italic><0.05）， indicating that the inflammatory model was successful. Compared with the normal group， the expression of TNF-<italic>α</italic> and COX-2 protein in the inflammatory model of RAW264.7 cells increased significantly（<italic>P</italic><0.05）， while the expression of TNF-<italic>α</italic> and COX-2 protein in bulleyaconitine A（BLA）， songorine， yunaconitine and karacoline groups decreased in varying degrees compared with the model group （<italic>P</italic><0.05）. Compared with the normal group， the expression of IRAK4， NF-<italic>κ</italic>B， JAK1 and STAT3 in the inflammatory model of RAW264.7 cells were significantly increased （<italic>P</italic><0.05）， while such levels in BulleyaconitineA， songorine， yunaconitine and Karacoline groups were significantly lower than those in the model group（<italic>P</italic><0.05）. Conclusion:Based on systematic pharmacology and <italic>in vitro</italic> experiments， the related targets and signal pathways were analyzed to provide new insights into the pathogenesis of RA， reveal the molecular mechanism of aconitum alkaloid in the treatment of RA， and provide new ideas for the application of Mongolian medicine in modern medicine.
ABSTRACT
Objective: To investigate the hydrolysis conversion rate of alcohol amine-diterpene alkaloids from aconitum alkaloids, hydrolyze aconitum alkaloids reference substance, calculate the amount of alcohol amine-diterpene alkaloids in the hydrolysis solution by the hydrolysis conversion rate, which is used as the amount of alcohol amine-diterpene alkaloids reference substance, and establish a content determination method for aconine, hypaconitine and aconine in Aconiti radix cocta.Methods: Through controlling the hydrolysis conditions of aconitine, hypaconitine and mesaconitine, aconine, hypaconitine and aconine were obtained.The determination was performed on an Agilent ZORBAX Extend-C18 RRHT(2.1 mm×50 mm,1.8 μm) column with the mobile phase consisting of methanol(A)-water(B containing 0.1% formic acid and 2.5 mmol·L-1 ammonium acetate) with gradient elution by HPLC-QTOF-MS.The flow rate was 0.21 ml·min-1.The column temperature was 30 ℃.MS instrument was equipped with an ESI+ ion source.Results: Under the hydrolysis conditions of this study, the conversion rate of aconine from aconitine was 99.64%;the conversion rate of hypaconitine from hypaconine was 99.94%;the conversion rate of mesaconitine from mesaconine was 99.57%.The HPLC-QTOF-MS methodological investigation showed the 3 kinds of alcohol amine-diterpene alkaloids were with good linearity (r>0.999 1).The RSD of the precision, repeatability and stability tests were less than 5%.The average recoveries were within the range of 99.43%-100.10%.Conclusion: The validated method is simple, specific, reliable and reproducible.In the absence of reference substance, it can be used for the quality control of the herbs of Aconitum L.species.
ABSTRACT
Objective:To develop a method of quantitative analysis of multi-components by single marker( QAMS) for nine kinds of alkaloids in Xiaohuoluo pills. Methods: An HPLC-QTOF-MS method with an Agilent ZORBAX Extend-C18 RRHT(2. 1 mm × 50 mm,1. 8 μm) column was applied. The flow rate was 0. 21 ml·min-1 . The column temperature was 30 ℃. The mobile phase was methanol (A)-water (B;containing 0. 1％ formic acid and 2. 5 mmol·L-1 ammonium acetate) with gradient elution. The aconitine was used as the internal standard, and the relative correction factor ( RCFs) of hypaconitine, mesaconitine, benzoylaconine, benzoyl-hypaconine, benzoylmesaconine, aconine, hypaconine and mesaconine was respectively established, and the reproducibility inspection on the RCF was performed. The contents of the other 8 kinds of aconitum alkaloids were calculated according to the RCF. At the same time, an external standard method ( ESM) was performed for the content determination of the nine alkaloids. The results of the two methods were compared. The feasibility and accuracy of the QAMS method were verified. Results:Within a certain range,the RCF of hypacontine,mesacontine, benzoylaconine, benzoylhypaconine, benzoyl mesaconine, aconine, hypaconine and mesaconine to aconitine was 1. 736,1. 979,1. 0471,0. 9242,1. 2901,1. 3078,1. 2859,and 1. 0948,respectively. The QAMS method was established for determi-ning alkaloids. There were no significant differences between the results of the QAMS method and those of the external standard method ( ESM) . Conclusion:With the validation of methodology, the method established in our study can be used for the content determina-tion of aconitine, hypaconitine, mesaconitine, benzoylaconine, benzoylhypaconine, benzoylmesaconine, aconine, hypaconine and mesaconine in xiaohuoluo pills.