| As a traditional Chinese medicine, Isatis indigotica Fort, is a clinical broad-spectrum antiviral medicament all this time. It is used extensively to treat influenza, mumps, epidemic encephalitis, hepatitis, etc. But its essence of clearing heat and removing toxicity and the material basis of antiviral activity is still indeterminate. In this paper, to study the main active substances of Isatis indigotica Fort., we start with protein, Polypeptide and amino acid, to study the main active substances of Isatis indigotica Fort.. We use modern separating and purifying technology and inspection methods to do extract, separation and identification. The main results were shown as follows:1. Study on extraction process of Isatis indigotica Fort. TPUse water extraction process, on the basis of single factor experiment, using extraction three factors and three levels response surface analysis (solvent, extraction time, solvent volume) to optimize the process conditions. Take Kjeldahl and BCA method to measure the total nitrogen (N) content and comprehensively analyze the results of the two different measurement methods.2. Content determination of four main amino acids in Isatis indigotica Fort.This is to determinate four kinds of mine amino acids in Isatis indigotica Fort, which come from different producing area by a pre-column derivatization HPLC method. Method:A pre-column derivatization HPLC method, in which the analyte was eluted in a Agilent ODS C18column (5μm,4.6mm×250mm) with phosphate-acetonitrile buffer with the flow rate1.0mL/min, was applied to analyze the content of Thr, Pro, arg and val. Conclusion:The method is efficient, sensitive and accurate. It can be used for the quality control of Isatis indigotica Fort.3. Study on degradation of Isatis indigotica Fort, in simulated gastrointestinal environment.Enzymatic reactions were terminated by adjusting the pH value of the solution. Concentration change of protein was detected by BCA. The result of degradation was described with SDS-PAGE gel electrophoresis, and the degraded state of small molecule substances was detected by HPLC. Result:Proteins of Isatis indigotica Fort, were mostly degraded in simulated gastrointestinal environment within180min, and transformed into low molecular peptide. A small amount of basic amino acid appeared in simulated intestinal fluid.4. Purification of Isatis indigotica Fort, peptide with cation exchange resinCation exchange resin001×14.5was chosen for purification of Isatis indigotica Fort, peptide from three different cation resins. The result of static adsorption performance showed that001×14.5had the best adsorbing ability of all these three. The elution process:the loading flow rate is1BV/h, after3minutes’standing, use2BV distilled water to wash the resin with a flow rate of2BV/h. Then use3BV pH9.5ammonia spirit as eluent to wash the resin at the same rate.5. Study on antiviral activities of Isatis indigotica Fort. Peptide①Death protection of Isatis indigotica Fort, peptide on H1N1(A/PR8/34) infected mice. The result indicates that Isatis indigotica Fort, peptide can improve the general condition of H1N1infected mice and protect the H1N1infected mice.②Study of Isatis indigotica Fort, peptide’s influence on the lung index number and pathology of the lung tissue. The result indicates that Isatis indigotica Fort, peptide can inhibit the replication of the H1N1in lung and improve the inflammation of the lung tissue.③Study of Isatis indigotica Fort, peptide’s influence on hyperplasy function of H1N1infected mice’s. The result shows that lymphocyte T and B’s replication function of the infectious model group is decreased compared with control group (P<0.01). Isatis indigotica Fort, peptide’s replication function of lymphocyte T and B are both significantly (P<0.01) higher in this two model group.④The result shows that Isatis indigotica Fort, peptide can improve the function of cell immunity and humoral immunity of H1N1infected virus. It is effective in inhibiting virus. |
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