Research Of The Potential Pharmacodynamic Ingredients Of Radix Isatidis And Its Mechanism

Radix Isatidis is the dry root of cruciferous plant Isatis indigotica,with heat-clearing and detoxifying effect,used in the treatment of various diseases caused by viral infection.At present,some progress has been made in the study of the material basis and the mechanism of Radix Isatidis,but the exact antiviral ingredients have not been found yet.The problems in this study are as follows:(1)in vitro research is more,but less in vivo.(2)the research of chemical constituents is complex,and the screening of antiviral related activities is not comprehensive.(3)there are many studies on simple anti-virus,but little research is done on the whole regulation.This paper aims at the above three issues related research,Which laid the foundation for the research of the material basis and the mechanism of Radix Isatidis(1)Study on serum pharmaceutical chemistry of Radix isatidis.The pretreatment and chromatographic conditions of plasma samples were investigated by HPLC and the fingerprints of plasma of Radix isatidis were established.Radix Isatidis plasma samples were compared with the blank plasma samples,it was preliminarily determined that there were 10 main components in the plasma containing Radix isatidis.The chemical constituents of Radix Isatidis aqueous solution,blank plasma and plasma were analyzed by LC-MS.Finally,59 chemical constituents were identified in the water extract of Radix Isatidis.Using XIC Manager function to check the drug-containing plasma samples for investigation and identification,20 kinds of chemical composition were found.According to the MS/MS spectrometry lysis and related literature reported,12 kinds of chemical composition were identified and 4 of them were compared with the standard to re-confirm.(2)Screening of active ingredients in Radix Isatidis.The chemical constituents of Radix Isatidis were screened by computer molecular docking technique,28 chemical constituents that may have inhibitory activity against neuraminidase,3 chemical constituents that may have inhibitory activity against hemagglutinin.(3’-hydroxyphenyl)-4(3H)-quinazolone,3-(2’-carboxyphenyl)-4(3H)-quinazolone,tryptone can be detected in plasma.High-throughput SPR successfully screened 13 compounds have inhibitory activity against NA,1 compounds have inhibitory activity against HA protein.The kinetic parameters and the affinity parameters of their interaction were obtained.The activity of neurospine was tested by fluorescence method with 4-MUNANA as the substrate.The results showed that two compounds had good inhibitory activity,eight compounds had weak inhibitory activity,three compounds had no inhibitory activity.The effect of indigo on inhibition of HA activity was confirmed by erythrocyte agglutination.(3)Study on antiviral mechanism of Radix Isatidis.The expression of RIG-I and TLR3-mediated antiviral innate immune signaling pathways in RAW264.7 cells infected by RSV/ploy(I:C)was studied by PCR,ELISA and Western Blot.The results showed that the transcription level and protein expression level of RIG-I and TLR3 signaling pathways were up-regulated in the cells when the cells were infected by virus,resulting in a high expression of IFN-β.The anti-viral mechanism of Radix Isatidis may down-regulate the phosphorylation of IRF3 by down-regulating the high expression of RIG-I and TLR3 signaling pathways induced by viral infection,and ultimately moderately inhibit the high expression of IFN-β induced by viral infection.The expression of IFN-β is maintained at a equilibrium point,which can both play an effective antiviral activity and reduce inflammation and tissue damage.