Analysis Of Components Absorbed Into Blood And Verification Of Anti-inflammatory Active Components Of Shuang Bei Tang Jiang And The Principal Drug Isatidis Radix In Vitro

Isatidis Radix is the dry root of cruciferae Isatis indigotica Fort.It is cold and bitter taste,used for external fever,the onset of febrile disease,sore throat,warm poison hair spot,erysipelas,carbuncle swollen sore poison.Isatidis Radix is the primal medicine of Shuang Bei Tang Jiang.Shuang Bei Tang Jiang is a compound preparation composed of 9traditional Chinese medicines,such as Isatidis Radix.It has a long history of clinical application and definite curative effect.However,there were few researches on its pharmacodynamic substance basis and effective components.In order to study the anti-inflammatory substance basis of Shuang Bei Tang Jiang,UHPLC-Q-Orbitrap HRMS analysis method was used to identify and compare the chemical components and components of the extract of Isatidis Radix and Shuang Bei Tang Jiang in vivo.The anti-inflammatory active components,core targets and mechanism of action of the primal medicine Isatidis Radix were studied by network pharmacology.The cytotoxicity of 11 components absorbed into blood was determined by MTT method,The inhibitory effect of11 components absorbed into blood on the release of inflammatory medium nitric oxide(NO)from mouse macrophages of RAW 264.7 induced by LPS was determined in the safe dose range of non-cytotoxicity,which could evaluate their anti-inflammatory activities.According to the results of MTT method and Griess method,the active ingredients with ideal NO activity were screened out.The inhibitory effects of 5 active ingredients,Isatidis Radix and Shuang Bei Tang Jiang on inflammatory factors TNF-α and IL-6 were further detected by ELISA experiment,and the main components of Isatidis Radix and Shuang Bei Tang Jiang on anti-inflammatory effects were discussed.It has certain significance to improve the quality control standard of Shuang Bei Tang Jiang,improve the study of its pharmacodynamic mechanism and guide its further clinical application.The chemical constituents of Isatidis Radix and Shuang Bei Tang Jiang were detected by UHPLC-Q-Orbitrap HRMS under positive and negative ion modes.A total of 71 chemical constituents were identified from water extract of Isatidis Radix and 66 chemical constituents were identified from Shuang Bei Tang Jiang.By comparison,malic acid,9(S),10(S),11(R)-trihydroxy-12(Z),15(Z)-octadecadienoic acid,hexocarbate,5-hydroxymethyl furfuric acid and indirubin could be identified in Isatidis Radix extract,but not in Shuang Bei Tang Jiang.The chemical components of Isatidis Radix and compound preparation Shuang Bei Tang Jiang were preliminarily identified,which laid the foundation for the study of the following components into blood and in vitro anti-inflammatory activity.Rats were given Isatidis Radix extract and Shuang Bei Tang Jiang by intragastric administration,and blood was collected from orbital venous plexus.The components in serum were analyzed by UHPLC-Q-Orbitrap HRMS.4’-hydroxy-7-methoxy isoflavones,5-hydroxymethyl furfuric acid,malic acid,hypoxanthine,2,5-dihydroxyindole,4-methoxy-2,3-indoledione,3-(2’-carboxyphenyl)-4(3H)quinazolone were only introduced into blood in Isatidis Radix extract group.Indigored,indoleacetamide,o-aminobenzoic acid-7-O-β-D-glucopyranoyl ester and eugenic acid could only be detected in the serum samples of Shuang Bei Tang Jiang group,and 38 common components might be the main components to exert the drug effect.The network pharmacological results showed that the anti-inflammatory active ingredients in Isatidis Radix mainly included 35 active such as brassicose glucosinosin,sericodontin,isovitexin and hydroxyindigo,33 potential anti-inflammatory targets of Isatidis Radix were mainly PTGS2,TNF,NOS2,MAPK14,etc.The pathways involved in achieving anti-inflammatory effects mainly include cancer signaling pathway,estrogen signaling pathway,thyroid hormone signaling pathway and neural active ligand-receptor interaction,etc.,which accorded with the characteristics of multi-component,multi-target and multi-pathway action of traditional Chinese medicine.The results showed that 5-hydroxymethylfurfural,isovitexin,hemicodontidine,cytidine and tryptamine,Isatidis Radix extract and Shuang Bei Tang Jiang had strong inhibitory effects on NO release from RAW 264.7 cells induced by LPS.Isovitexin,hemicodontidine and tryptamine inhibited the release of TNF-α and IL-6 from RAW 264.7macrophages induced by LPS.Isatidis Radix extract and Shuang Bei Tang Jiang could significantly inhibit the release of TNF-α and IL-6 in a good dose-dependent manner.In conclusion,the anti-inflammatory components isovitexin and semisodontidine obtained by network pharmacological screening of Isatidis Radix,could be detected in serum samples of rats administered with Isatidis Radix and Shuang Bei Tang Jiang group,showing good anti-inflammatory activity.Isovitexin,tryptamine and semisodentine were the key anti-inflammatory active components of Isatidis Radix and Shuang Bei Tang Jiang.