| Objective:The current quality evaluation methods of Anemarrhena asphodeloides and salted Anemarrhena asphodeloides are mainly based on the determination of chemical components.This project intends to establish a quality evaluation method of Anemarrhena asphodeloides based on biological activity,so as to provide a reference for more scientific and reasonable evaluation of the quality of Anemarrhena asphodeloides.Methods:1.Using the method of network pharmacology,the chemical components and inflammatory targets of Anemarrhena asphodeloides were constructed into a "component target disease" network,and then the topological parameters of the network were analyzed to obtain the main anti-inflammatory protein targets of Anemarrhena asphodeloides,and then the anti-inflammatory pathway enrichment analysis of Anemarrhena asphodeloides was carried out.2.Cyclooxygenase-2 inhibitor screening kit(COX-2 inhibitor screening kit)was used to determine the biological activities of nine chemical components and celecoxib in Anemarrhena asphodeloides.Molecular docking technology was used to docking the high active components of Anemarrhena asphodeloides with COX-2 protein targets to evaluate their interaction mode;The inhibition rate of Rhizoma Anemarrhenae against COX-2 before and after processing was determined,and the quality biological activity evaluation method based on COX-2 inhibition was established.3.9 chemical components in Anemarrhena asphodeloides were identified by UPLC-Q-TOF-MS / Ms.the fingerprints of nine active components in Anemarrhena asphodeloides were established and the similarity was calculated;The content changes of 9 active components before and after processing were compared,and the change rule of chemical components before and after processing was evaluated;The spectrum effect correlation analysis of chemical components and biological activities of Anemarrhena asphodeloides before and after processing was carried out by metaboanalyst online analysis tool,and the chemical components closely related to anti-inflammatory activity were screened.The pharmacodynamic weight of each component was calculated by the chemical component content and biological activity of Anemarrhena asphodeloides,and the effective component index based on mangiferin was established.Results:1.Through the network pharmacology research,we obtained 30 active components of Anemarrhena asphodeloides,and predicted 34 main anti-inflammatory protein targets of Anemarrhena asphodeloides,of which the main protein target is COX-2.The results of enrichment analysis of KEGG and go pathway showed that the anti-inflammatory activity of Anemarrhena was mainly related to MAPK signaling pathway,tumor necrosis factor signaling pathway,I-κ B-kinase / NF-κ B signal and T cell receptor signal pathway.2.The inhibitory effect of nine monomers of Anemarrhena asphodeloides on COX-2 activity was determined.It was found that mangiferin had strong inhibitory activity,which was close to that of celecoxib;Through the molecular docking of mangiferin with COX-2 protein target,the results showed that mangiferin acted through a new compound binding site on COX-2 target,which was significantly different from the traditional celecoxib binding site.After 15 batches of Anemarrhena asperata were processed,the effect of inhibiting COX-2 activity was enhanced(P<0.05).A quality evaluation method based on the effect of inhibiting COX-2 activity was established.The methodological investigation results showed that the relative standard deviation of precision(RSD,The relative standard deviation)is 3.88%,and the repeatability RSD value is 12.45%.3.Through fingerprint study of Anemarrhenae,it was found that the similarity of each batch was greater than 0.9 except 02 batches,and the similarity was better;The content of mangiferin,anemarrhenosin AII and anemarrhenosin AIII increased(P <0.05),while the contents of neomangiferin,saponin BII and saponin E decreased(P <0.05),while the contents of anemarrhenosin AI and I were not significantly changed(P > 0.05);The correlation analysis of chemical fingerprint and biological activity showed that mangiferin had the strongest correlation with anti-inflammatory activity in Anemarrhena asphodeloides;The pharmacodynamic weight of mangiferin was 0.97326,and the effective component index of mangiferin was successfully constructed.Conclusion:In this study,the main anti-inflammatory target of Anemarrhena asphodeloides was predicted to be COX-2 by network pharmacology.The main active component of Anemarrhena asphodeloides was mangiferin by UPLC-MS / MS and correlation analysis of chemical fingerprint and biological activity;Molecular docking showed that mangiferin acted through a new binding site;The biological activity evaluation method of Anemarrhena asphodeloides against COX-2 was established.The changes of biological activity and chemical components before and after processing were analyzed.The effective component index of mangiferin was established.It is proved that mangiferin is the active component of Anemarrhena asphodeloides in inhibiting the activity of COX-2 in the quality control of Anemarrhena asphodeloides《 The quality control range of mangiferin before and after processing of Anemarrhena asphodeloides stipulated in Chinese Pharmacopoeia 2020 edition is unreasonable,which needs to be further explored.This study provides technical support for more scientific and reasonable evaluation of the quality of Anemarrhena asphodeloides,and provides reference for clinical rational drug use and new drug development of Anemarrhena asphodeloides. |
