| As a traditional Chinese medicine,Radix Isatidis is derived from the dried roots of the crucifer plant Isatis indigotica Fort.By clearing heat and detoxifying cold blood or pharynx,it is mainly used to treat bacterial infections and viral diseases,including influenza virus,hepatitis B virus,RSV,herpes simplex virus,mumps virus,coxsackie virus,etc.Meanwhile,it is commonly considered as a broad-spectrum natural antiviral drug.We have previously found that there is a synergistic anti-virus effect of different fractions from Radix Isatidis,but it is unkown which components are used or what kind of mechanism works in Radix Isatidis.In view of problems in the above research,we focuses on the antiviral active components of Radix Isatidis and studies the mechanism of antiviral synergy around its intervention on the immune signaling pathway.The specific contents include the following aspects:(1)Study of preparation of effective parts of Radix IsatidisBased on the orthogonal design,the extraction process of total alkaloids,total lignans and total organic acids from Radix Isatidis was investigated,and the optimal extraction process of three parts from Radix Isatidis was optimized.The macroporous resin adsorption and ethyl acetate extraction were used to prepare the total alkaloids.In the separation process,more than three kinds of alkaloid precipitation reagents were combined for detection.The fractions positive for the three reagents simultaneously were selectively collected to further remove other types of impurities.To detect alkaloid contents,indole-3-carbaldehyde was employed as a reference substance(alkaloids in Radix Isatidis are mainly indoles).The sample was first reacted with bromocresol green,and then an appropriate range of color change was selected for measurement using the characteristic wavelengths of the products,giving more accurate results.The purity of total alkaloids of Radix Isatidis was 50.03%by ultraviolet spectrophotometry.This study used UV spectrometry based on the similar UV absorptions of molecules with the same parent nucleus while using macroporous resin to adsorb and to enrich lignans.Taking larch resin alcohol,a representative lignan component,as the reference substance,various streams were tested by the whole-wavelength scanning,and the lignan components which had consistent UV spectra with that of the reference substance were collected by segmentation,aiming to ensure the fraction had a similar chemical structure to that of control.The purity of the total lignans from Radix Isatidis was 74.87%.The organic acid fraction from Radix Isatidis was extracted by ethyl acetate.The solution was passed through an SA-2 cationic resin column to remove amino acids,and the filtrate was collected.The solution was tested for ninhydrin reaction before and after filtration until the result was not positive.The filtrate was concentrated under reduced pressure and dried into an organic acid sample.The total organic acid(BO)content marked by salicylic acid was determined by potentiometric titration to be 74.81%.Then,HPLC method was used to identify the main components of the three fractions from Radix Isatidis.9 of which were identified by comparing the retention times with those of standard reference compounds,i.e.Salicylic acid,Epigoitrin,Isatin,Oxindole,Indole-3-carboxaldehyde,Lariciresinol-4,4'-bis-O-?-D-glucopyranoside,3-Indoleacetonitrile,Lariciresinol,1-Methoxyindoleacetonitrile.It provides quality assurance for subsequent pharmacological activities.(2)In vitro and in vivo antiviral effects of different effective fractions of Radix IsatidisThe cell model was established by virus infection,and then the drug was performed to interven,and the synergistic index of the combination drug was evaluated by using calcusyn software.When the three sites are combined,the antiviral effect is stronger than that of the various fractions from Radix Isatidis alone,which is slightly weaker than the ribavirin-positive drag.When the concentration was less than 100?g/mL,CI<1,indicating that the three effective fractions from Radix Isatidis have a synergistic effect on the anti-RSV virus.For the in vivo efficacy experiment,the mice were treated with different effective fractions per se or in combination from Radix Isatidis,and an animal model was established by intranasal infection to observe the changes of interferon P and inflammatory factors in the lung injury and alveolar lavage fluid of the mice.The study found that after the treatment of combination of the three fractions,the degree of lung injury in mice can be well protected and the high expression of interferon(3 and inflammatory factors can be inhibited.The effect of the three-site high-dose group is not different from that of ribavirin-positive drugs.(3)Synergistic antiviral mechanism of different effective fractions of Radix IsatidisGene chip technology was used to analyze differential genes before and after drug action and to screen potential targets for possible action.Through the expression trends of the molecules in the classical pathway and the classical pathway enrichment analysis statistics,we found and mapped the relevant signal pathways.In the previous study,we have found that Radix Isatidis is able to regulate the TLR3 pathway,so we focus on the RIG-I/MDA5 signaling pathway and JAK/STAT signaling pathways containing MAVS cross-regulatory points.PCR and Western Blot were used to detect the expression level of related mRNA and protein in the signaling pathway.The results showed that the combination of total organic acids,lignans and alkaloids from Radix Isatidis was more effective than those of individual treatments.Organic acids,lignans and alkaloids have different regulatory effects on each key molecule in the pathway.In the RIG-I/MDA5 signaling pathway,lignans have a stronger down-regulation effect on P-IRF3,while alkaloids have a stronger down-regulation effect on MDA5,indicating that each effective fraction from Radix Isatidis could inhibit the RSV through regulating different key molecule in the pathway.In the JAK/STAT signaling pathway,the study found that the active sites from Radix Isatidis have a regulatory effect on the JAK1/STAT1 signaling pathway,and there is a synergistic regulation effect in the three fractions from Radix Isatidis.The combination of three fractions from Radix Isatidis has a stronger down-regulation effect on both STAT1 and SOCS1 molecules regardless on the mRNA level or the protein expression level.However,the results showed that the total organic acids,total lignans and total alkaloids from Radix Isatidis have stronger regulation effect on JAK1 molecules than that of the combination of three fractions.This result suggests that the active components of organic acids,lignans and alkaloids from Radix Isatidis may act upstream and downstream of JAK1 molecular targets,which also confirms the multi-component and multi-target characteristics of traditional Chinese medicine. |
PDF Full Text Request