Quality Consistency Assessment Of Root And Stem Of Zanthoxylum Nitidum Based On Anti-inflammatory Activity

Objective:A quality evaluation method of Zanthoxylum nitidum(ZAN)based on multi-component chemical characterization and biopotency determination was established.The quality of root and stem of ZAN was further evaluated from the perspective of quality biological evaluation,in order to provide more reference for comprehensive development and utilization of ZAN resources.Method:1.Multicomponent Rapid Chemical Characterization Analysis of Roots and Stems of ZANIn this study,20 batches of ZAN medicines from different origins(including root and stem)were collected,and UPLC method was used to construct fingerprints of root and stem of ZAN.The similarity between root and stem of 20 batches of ZAN medicines was evaluated by similarity evaluation software of Chinese medicines(2004 edition).A method for determination of nine components in the root and stem of ZAN was established.The contents of the components were determined,and the components of the root and stem of ZAN were comprehensively analyzed by cluster analysis,principal component analysis and least squares discriminant method.2.Anti-inflammatory Targets and Mechanism Prediction of ZAN Based on Network PharmacologyThrough pharmacological analysis platform(TCMSP)database of TCM system and Pharmmapper server at home and abroad,using oral availability(OB%)and drug-like properties(DL)as restrictive conditions,the components of ZAN were screened,predicted and collected related targets.Using OMIM and other databases to screen genes and protein targets related to inflammation,STRING database to construct interaction network between inflammation targets,PPI analysis and construction of "active ingredients-inflammation targets" network,and obtain potential targets directly related to ZAN intervention in inflammation,KEGG pathway enrichment analysis to screen out pathways closely and significantly related to inflammation,and build "activity Component-Inflammatory Target-Anti-Inflammatory Pathway" Network was used to screen out potential targets and their mechanisms closely related to the anti-inflammation effect of ZAN,in order to find a reference for the selection of biological assay for quality evaluation indicators of ZAN.3.Study on Anti-inflammatory Mechanism of ZAN Based on TranscriptomicsStudy on Anti-inflammatory Mechanism of ZAN Based on Transcriptomics.In this chapter,RAW264.7 mouse macrophages associated with inflammation were selected as the research objects.Transcriptional histology was used to investigate the whole gene expression of LPS-induced inflammatory macrophages treated with ZAN.The differential expression genes and important connections between inflammatory cell group and inflammatory model group were screened by canonical correlation and KEGG pathway analysis.Path and key targets were further validated by animal experiments.The results of this study can be used to verify the prediction of anti-inflammatory mechanism of ZAN by network pharmacology on the one hand;and secondly,it can be used to screen indicators closely related to the mechanism of action for quality evaluation,laying a foundation for the selection of biological assay for quality control indicators of ZAN.4.Study on Biological Assay Method for the Consistency of Root and Stem Quality of ZANStudy on Biological Assassy Method for the Consistency of Root and Stem Quality of ZAN Taking the key target screened from the above studies as the biological assay for quality control indicators,a rapid,sensitive,stable and reliable method for determination was selected and the methodology of the method was investigated,so as to establish a biopotency determination method of ZAN based on anti-inflammatory activity.The established biopotency determination method was used to evaluate the sample preparation methods for biological assay for quality control of ZAN.On the basis of single factor investigation,combined with star design-effect surface method,the optimum extraction scheme was screened.The optimum sample preparation method and established biopotency determination method were used to prepare and determine the anti-inflammatory activity potency of root and stem samples of ZAN.The anti-inflammatory activity potency of root and stem samples of ZAN in different batches were obtained.The potency of the two samples were compared and their anti-inflammatory effects were evaluated.On the basis of the quality consistency,the components which have great influence on the activity of bioassay indexes are preliminarily screened by correlation analysis of chemical fingerprints in the previous chapters.Finally,molecular docking technology is used to explore the binding mode between components and targets,in order to explore the possible material basis of influencing the activity of biological assay for quality control indicators.Results1.Multicomponent Rapid Chemical Characterization Analysis of Roots and Stems of ZANThe similarity of fingerprints in the roots of ZAN is mostly greater than 0.8,while the similarity of fingerprints in the stems is lower than 0.8.The chemical constituents in the roots and stems of Acanthopanax bidentata are basically the same,and the contents of flavonoid glycosides and lignins are the same.Some constituents are different,especially chelerythrine,magnoflorine and nitidine chloride and other alkaloids;Cluster analysis and principal component analysis roughly divided the root and stem of 20 ZAN into two categories,in which there were five batches of ZAN root and all batches of its stem clustered into one group;Least Partial Square Discriminant Analysis clearly distinguished the root and stem of ZAN,and the main components causing the difference between them were chelerythrine,magnoflorine and nitidine chloride.2.Anti-inflammatory Targets and Mechanism Prediction of ZAN Based on Network PharmacologyNine potential targets directly related to the anti-inflammatory effects of ZAN were screened and 16 significant signaling pathways were obtained by enrichment analysis of these targets.TNF signaling pathway is closely related to inflammation and is the most significant one.It may be the main way for ZAN to exert anti-inflammatory effects.As the downstream target of this pathway,COX-2 has a large topological parameter and can be used in many ways.Therefore,COX-2 plays a key potential role in the anti-inflammatory activity of ZAN.As a phenotypic factor,COX-2 can be used to reflect the anti-inflammatory effect of the ZAN and provide a reference for the selection of indicators for the biological evaluation of the quality of ZAN.3.Study on Anti-inflammatory Mechanism of ZAN Based on TranscriptomicsZAN mainly exerts its anti-inflammatory effect by interfering with TNF-NF-KB-Toll-like signaling pathway.The expression of COX-2,IL-6 and TNF-αdownstream targets in this pathway is also significantly down-regulated.Animal experiments of inflammatory model also show that ZAN can significantly downregulate and reduce the expression of COX-2 and the content of PGE2 and IL-6.4.Study on Biological Assay Method for the Consistency of Root and Stem Quality of ZANWith COX-2 as the biological evaluation index,a method for the determination of COX-2 inhibitory activity potency of ZAN was established.The methodological and sample preparation methods of this method were investigated and optimized.The results showed that the method for the evaluation of biological activity of ZAN had high precision and good repeatability,and the corresponding sample preparation method was stable,reliable and operable.The results of COX-2 inhibitory activity potency showed that:ZAN had high precision and good reproducibility.The overall COX-2 inhibitory activity of root and stem was similar,and the overall potency of root and stem mixed samples was higher than that of both.The screening results of the active substance basis for COX-2 inhibitory activity showed that not only alkaloids such as chelerythrine,but also flavonoid glycosides such as hesperidin and diosmin,lignans such as L-sesamin and L-asarinin existed in the components of ZAN.It has certain COX-2 inhibitory activity,especially chelerythrine,L-sesamin and L-asarinin,which have great contribution to the inhibition of COX-2 activity by ZAN.Components with high correlation with COX-2 inhibitory activity include diosmin,hesperidin,nitidine chloride,chelerythrine,L-sesamin and L-asarinin.The order of their inhibitory effect on COX-2 activity is L-asarinin>L-sesamin>chelerythrine>hesperidin>diosmin>nitidine chloride.The contribution order of monomers to the inhibition of COX-2 activity is in accordance with the order of monomers.The second was L-sesamin>chelerythrine>L-asarinin>nitidine chloride>hesperidin>diosmin.The results of molecular docking with COX-2 showed that L-asarinin had the most affinity to COX-2,followed by L-sesamin,and finally chelerythrine.The results were consistent with their potency.Conclusion:The quality biological evaluation method established in this study can provide reference for the improvement of quality control standard of ZAN,and further prove that the anti-inflammatory activity of root and stem of ZAN is similar from the point of view of quality biological evaluation.In anti-inflammatory aspect,the stem of ZAN has potential application value of medication with its root.