Study On The In Vivo Medicinal Chemistry And Anti-Inflammatory Molecular Targets Of Lignans From Isatidis Radix

Isatidis Radix is a dried root of the cruciferous plant Isatis indigotica Fort,which has the functions of heat-clearing and detoxification,cooling blood and benefiting pharynx.The main treatment is exogenous fever,warm disease early,swollen sore throats,epidemictyphus,erysipelas,carbuncle swollen chuangdu.Isatidis Radix,as a typical heat-clearing and detoxifying drug,has antiviral,anti-inflammatory,antioxidant and other pharmacological effects.As one of the main chemical components in medicinal materials,lignans have antioxidant,anti-inflammatory,immune regulation and other activities,which are closely related to the antiviral effect of isatidis Radix.However,the specific in vivo medicinal ingredients and targets are not clear.Therefore,this topic revolves around this type of component and carries out the following research work:(1)Study on the chemical constituents of lignans in Isatidis RadixThe Isatidis Radix is extracted three times with 8 times the amount of 80%ethanol.The extracts are combined to recover the ethanol.After extraction with n-butanol,the n-butanol is extracted to obtain the extract.Using D101 macroporous adsorption resin,ODS reverse chromatographic column,high performance liquid chromatography,Flash analysis and purification,semi-preparative liquid chromatography and other chromatographic separation methods,the components of the n-butanol part of Isatidis Radix were separated.Then the chemical properties and NMR data of the compounds were combined to identify the structures of the compounds.A total of 11 chemical components were isolated,including 3 lignans,2 simple phenylpropenes,2 flavonoids,2 alkaloids,1 nucleoside,and 1 amino acid.(2)Determination of lignans in Isatidis RadixThe extract obtained from Isatidis Radix was extracted with 80%ethanol and eluted with gradient ethanol of different concentrations through D101 macroporous adsorption resin for partial collection.Application of ultraviolet visible wavelength scanning,according to the characteristic absorption detection,merge collect get total wooden fat element components parts.Uv spectrophotometry was used to determine the content of 67.85%of total lignans in Isatidis Radix with Lariciresinol-4-O-β-D-glucopyranoside as control.The fingerprint of total lignans of Isatillanum isatillanum was obtained by high performance liquid chromatography(HPLC).The retention time of the reference substance in the HPLC spectrum was combined with uv full-wavelength scanning for qualitative analysis,and the content of each monomer component in total lignans was calculated.Finally,the content of three lignans isolated from total lignans of Isatidis Radix was 23.99%.(3)pharmacochemical study of total lignan serum from Isatis root.The total lignans of Isatidis Radix were administered to rats by gavage,and blood was collected from the abdominal aorta to obtain serum.Establish the serum medicinal chemistry map of Isatidis Radix total lignans by HPLC and use LC-MS(liquid chromatography-mass spectrometry technology)to process the data with Peakviewl.2 software to identify the total lignans into the blood components and their metabolites.Finally,a total of 25 chemical components were identified in the total lignin samples of Isatidis Radix root.16 compounds were identified in the drug-containing serum,including 7 prototype components and 9 metabolites.Network pharmacology study on inflammation of lignan in Isatidis Radix(4)Network pharmacology study on inflammation of lignans from Isatidis RadixIn the TCMSP database,the compound name and CAS number of the isatis lignan component are used to search for the component and its target protein name,and the target name is converted into a gene symbol on the STRING network.In the TDD database,DisGeNET database,and GeneCards three databases,"Inflammation" is used as a search term to search for disease targets,remove irrelevant proteins,and obtain disease-related target proteins.The intersection of component target and disease target was obtained by using wynn,and protein interaction analysis of drug-disease target genes was performed in the STRING network.GO enrichment analysis and KEGG analysis were performed on the PPI network screening results,and signal pathways were screened according to Pvalue value.Then,the component target pathway network was plotted in Cytoscape software.In the end,24 components and disease intersection targets were screened.The more relevant targets are PTGS2(COX-2),HSP90AA1,NCOA2,PPARG,etc.The main pathways involved are cancer pathway,estrogen signaling pathway,breast cancer,prostate cancer,thyroid hormone signaling pathway,regulation of lipolysis in fat cells,IL-17 signaling pathway,arachidonic acid metabolism,NF-κB signaling pathway,serum Prime synapse.(5)Screening and in vitro validation of COX-2 inhibitorsAutoDock Vina software was used to perform molecular docking between components and target COX-2,and target was selected based on the results of network pharmacology research.Celecoxib was used as the control drug according to literature review.The results of molecular docking showed that 7 compounds,including 2-Methoxy-4-vinylphenol_qt,lariciresinol-4,4’bis-O-β-D-glucopyranoside,Lariciresinol-4’-O-β-D-glucopyranoside,Lariciresinol-4-O-β-Dglucopyranoside,Pinoresinol,clemastanin B_qt,and Medioresil were superior to the control.It is proved that the predicted components have a great possibility to combine with the key targets.The results showed that the lignin component of Isatidis could play an anti-inflammatory effect by regulating COX-2.According to the results of molecular docking,the representative lignan components of Isatidis Radix,Lariciresinol,lariciresinol-4,4’-bis-O-β-D-glucopyranoside,Lariciresinol-4-O-β-D-glucopyranoside inhibition effect on COX-2.The results showed that the inhibitory effects of Lariciresinol and Lariciresinol-4-O-β-D-glucopyranoside on COX-2 were significant,and their IC50 values were 1.41 μM,2.01 μM.