Studies On The Chemical Compositions And The Anti-Inflammatory Action Mechanism Of The Compound Angelica Injection

Objective:Compound Angelica Injection?CAI?,composed of Angelicae Sinensis Radix?Ligusticum Chuanxiong and Carthamus tinctorius L,has the effect of Huoxue Tongluo and Quyu Zhitong.In recent years,it is widely clinically used in the treatment of cardiovascular diseases,but its active ingredients and Action mechanism need to be further research.In this paper,the simultaneous content detection method of main chemical components and multi-compounds from CAI and the molecular mechanism for the treatment of cardiovascular diseases were evaluated by using chemical analysis,network pharmacology and molecular biology techniques.Methods:1.An UPLC-Q-Orbitrap MS/MS method was developed for identification of the chemical constituents in CAI.The constituents from CAI were identified based on the precise molecular weight,the secondary mass spectrometry and the retention time.A HPLC-DAD method was established to simultaneously determine the contents of multi-target ingredients in CAI.2.The identified chemical components of CAI and corresponding targets were obtained through Traditional Chinese Medicine Systems Pharmacology Database?TCMSP?and Swiss Target Prediction website.OMIM,TTD and PharmGkb were used to predict and screen the targets of CAI.The protein interaction network was constructed using the Cytoscape3.2.1 software.Gene function analysis?GO?and enrichment analysis based on the Kyoto Encyclopedia of Genes and Genomes?KEGG?pathway involved in the targets were analyzed by DAVID6.8.The effectiveness of CAI were validated on the cell level.3.LPS-induced RAW264.7 cells inflammation model was established in vitro.The cells were divided into control group,model group?LPS?,CAI treatment group(respectively treated with 2.5?5 and 10?L·mL^-1 CAI 12 h,next stimulated 12 h by LPS)and the Cryptochlorogenic acid treatment group?respectively treated with 100?200 and 400?mol Cryptochlorogenic acid 12 h,next stimulated 12 h by LPS?.Griess reagent assay was used to detect NO content in cell supernatant,the transcription levels of iNOS?COX-2?IL-6?TNF-?and IL-10 were detected by RT-PCR and the expressions of iNOS?COX-2 and HO-1 was detected by Western Blot.After using HO-1 specific inhibitor ZnPP to block the expression of HO-1 in the cells,Griess reagent assay was used to detect NO content in cell supernatant of LPS-induced RAW264.7 cells.Results:1.Based on the precise molecular weight,secondary mass spectrometry and retention time,six compounds in CAI extract were identified or tentatively characterized,including Neochlorogenic acid?Chlorogenic acid?Cryptochlorogenic acid?Caffeic acid?Hydroxysafflor yellow A and Ferulic acid.The HPLC-DAD method was established to simultaneously determine the contents of six components.Using this method,the quality of CAI and 14 batches of commercially available CAI were evaluated.This established method could be utilized as a quality control method for CAI with high stability and accuracy.2.Six components including Neochlorogenic acid?Chlorogenic acid?Cryptochlorogenic acid?Caffeic acid?Hydroxysafflor yellow A and Ferulic acid were confirmed by Pubchem database and about 589 targets of six components were obtained from TCMSP and Swiss Target Prediction website.767 targets related with the CVD were collected from OMIM,TTD and PharmGkb database.The protein interaction between the component targets of CAI and the targets related to cardiovascular disease was analyzed by STRING database.Cytoscape3.2.1 software was used to visualize the interaction between drug-related targets and disease-related targets.The Network Analyzer plug-in was used to analyze the Network topological properties of each node in the Network and to select the targets above the mean as the key targets.The key target of CAI for the treatment of CVD were selected,mainly including IL-1??PTGS2?STAT3 and TLR4.KEGG pathway enrichment showed that the main pathways of CAI in the treatment of CVD were Toll-like receptor signaling pathway and TNF signaling pathway,suggesting that CAI might regulate inflammatory response,which may be one of the mechanisms of its treatment of CVD.The anti-inflammatory substance?NO?scavenging based on the RAW264.7 cell model was established.The result of spectrum-effect relationship showed that Cryptochlorogenic acid?Hydroxysafflor yellow A?Caffeic acid?Neochlorogenic acid?Chlorogenic acid and Ferulic acid were the main anti-inflammatory ingredients of CAI.3.CAI and cryptochlorogenic acid can significantly inhibit the production of NO,the mRNA expression of iNOS,COX-2,IL-6 and TNF-?and the expression iNOS?COX-2 and promote the expression of IL-10.CAI and cryptochlorogenic acid can significantly induce the expression of HO-1 protein in a concentration-dependent manner and the inhibition of CAI and cryptochlorogenic acid on the production of NO in the supernatant of RAW264.7 cells induced by LPS could be partially reversed by ZnPP,suggesting that CAI and cryptochlorogenic acid could inhibit LPS-induced inflammatory in RAW264.7 cells via HO-1 induction.Conclusion:Six constituents were identified from CAI using UPLC-Q-Orbitrap MS/MS.A HPLC-DAD method was developed and validated for simultaneously determination of six constituents.The results of network pharmacology showed that inflammatory response plays an important role in the treatment of CVD.The result of spectrum-effect relationship showed that cryptochlorogenic acid?Hydroxysafflor yellow A?Caffeic acid?Neochlorogenic acid?Chlorogenic acid and Ferulic acid were the key anti-inflammatory ingredients of CAI.LPS-induced RAW264.7 cells inflammation model was established to study the preliminary mechanism of anti-inflammatory effect of CAI and cryptochlorogenic acid.This research will lay a material foundation for further research on the quality control,mechanism of action and the secondary development ideas of CAI and provide a new ideas and research method for the study of the mechanism of Chinese herbal compound.