Antiviral Activity Of Folium Isatidis Against Respiratory Viruses Infection

Folium Isatidis is one of the traditional Chinese medicine derived from the cruciferous plants woad (Isatis tinctoria L.) dried leaves. The main chemical composition contains indigotin, indirubin,3-hydroxybutyrate phenyl quinazolinone, tryptanthrin, organic acids, etc. The antiviral effect of Folium Isatidis has been recorded in the clinical treatment of influenza, bronchitis, encephalitis, and mumps and other diseases. In this study, antiviral activity of the four extracts derived from the Chinese medicine Folium Isatidis was observed in vitro and in vivo.Objective:A series of four fractions (Fraction Ⅰ,Ⅱ,Ⅲ,Ⅳ) from Folium Isatidis were isolated. The antiviral effect of the four extract against a panel of RNA and DNA viruses in vitro, namely influenza A virus (IAV), respiratory syncytial virus (RSV), coxsackie virus B3(CVB3) and adenovirus type7(Ad7) were investigated. The influenza virus infected pneumonia mice model were established at the same time to investigate the antiviral effect and possible mechanisms of Folium Isatidis extract (Fraction III) in vivo.Methods:Experiment was divided into two phases. The first stage is to investigate the in vitro antiviral activity of four Folium Isatidis extracts (Fraction Ⅰ,Ⅱ,Ⅲ,Ⅳ) and their mechanism of action. This process was investigated based on three ways of action: virus growth inhibition assay, virucidal assay, treatment of extracts before virus infection assay to detect inhibiting effect of viruses like IAV, CVB3, RSV and Ad7. Four extracts toxicities were also evaluated by MTT assay on canine kidney (MDCK) or human laryngeal epithelial (HEp-2) cells. Observation of inhibition of IAV, RSV, Ad7and CVB3-induced cytopathic effect (CPE), MTT assay, plaque reduction test (PRA) were used to evaluate the antiviral effect of four Folium Isatidis extracts against IAV, RSV, CVB3and Ad7. The mice infected with influenza A virus pneumonia were used to evaluate the in vivo antiviral effect of Folium Isatidis extracts (Fraction Ⅲ). Mice were treated with the indicated doses (200mg/kg/d,100mg/kg/d,50mg/kg/d) of Folium Isatidis fraction Ⅲ for5consecutive days postinfection. Survival rate and weight reduction was calculated from their original weight on day0. The lung index (the ratio of mean lung weight to mean body weight) was used to evaluate lung inflammation. Levels of influenza viral M gene RNA were determined by the qPCR assay. Lung inflammation was evaluated by observing the mouse lung tissue biopsy.Results:1. The four extracts from Folium Isatidis exert wide spread antiviral effect against RSV, Ad7, CVB3, IAV. The TD50of Folium Isatidis fraction III for HEp-2cells was57.09±2.92; The TD50of Folium Isatidis fraction I, II for HEp-2cells were23.15±1.49,18.9±2.01μg/ml, respectively. No significant cytotoxicity could be observed on HEp-2and MDCK cells at concentrations lower than15μg/ml of fractions I and II. Among the four fractions obtained, fractions I and II were the most toxic on HEp-2and MDCK cells.2. Through three different mode of actions, four Folium Isatidis extracts showed strong inhibitory when adding after virus penetration. It was found as the dose of the drugs increased, the degree of CPE decreased but the viral inhibition rate increased. Among the four fractions extracted, fractions III and IV from Folium Isatidis were the most effective and could completely inhibit a wide range of viral replication. The selectivity index of fractions III when used against IAV was higher than others. No direct virucidal activity of each fraction was observed. Also no inhibitory activity was observed when different cells were pre-incubated with various concentrations of four fractions.3. Mice challenged with10LD50influenza A virus (FM1strain) were orally administered with200,100or50mg/kg/d fraction III, respectively. Compared with the virus control group, the Folium Isatidis fraction III can significantly reduce the mortality of viral pneumonia in mice (P<0.01), prolonged the mean survival time (P <0.01), to improve the survival rate of mice infected with the virus. Fraction III also caused a dose-and time-dependent reduction in the lung index (lung weight/body weight) at3,6and9days post-infection. The vRNA in the lung decreased significantly in the fraction Ⅲ-treated group (200mg/kg/d). Therefore, Folium Isatidis fraction III can exert strong antiviral effects in viral replication, accompanied by prolonged survival rate, attenuated pulmonary virus titers and lung index.4. In vivo, Folium Isatidis fraction Ⅲ can alleviate the pathological lesion caused by lethal influenza virus infection in mice. The severity of viral pneumonia was significantly reduced with the increase the dosage. Histological examination of mice treated with low dose of Folium Isatidis fraction Ⅲ and viral control group had obvious thickened alveolar walls, peri-bronchial inflammation, prominent bronchial infiltration of macrophage and neutrophils in the alveolar walls, interstitial lymphocyte infiltration, even severe hemorrhage.Conclusion:1. The four extracts from Folium Isatidis exert good antiviral effect against IAV, RSV, Ad7, CVB3infection. Its mode of actions was primarily based on affecting virus propagation after viral penetration.2. In mouse study, Oral administration of200mg/kg/d of Folium Isatidis fraction III exerted strong antiviral effects in viral replication, accompanied by prolonged survival rate, attenuated lung tissue damage as well as significant reductions in pulmonary virus titers and lung index.