| ObjectiveIn order to study the anti-H1N1influenza virus activity of Compound Longqincao, the solubilization and trapping effects of Microemulsion drug-carrier system for a variety of active ingredients in many Compounds, the microemulsion extract of compound Longqincao (MLQC) has been prepared via extracting Compound Longqincao by using Biocompitible Microemulsion (ME) which was designed in our pre-expariements. On the basis of preliminary experiments of MLQC containing higher contents of baicalin, liquiritin and amino acids, the antiviral effects of this compound on H1N1 influenza virus in vitro has been studied comprehensively. The main components have been selected for anti-virus test, and the anti-H1N1 influenza virus activities in the prescription were excavated preliminary. As to find prescription ratio with the best anti-viral effect, the combinations were optimized by using uniform design, and the therapeutic basis of MLQC was excavated at the same time. The preliminary antiviral mechanisms of MLQC and ME have been explored via constructing component-protein-target pharmacology network.Methods2.1 To study the anti-H1N1 influenza virus effects of MLQCThe MLQC was obtained by extracting Compound Longqincao with ME as solvent. The maximum non-toxic concentration (TCo) and median toxic concentration (TC50) of ME and MLQC on A549 cells were detected by using MTT. The influences of these drugs on virus-induced cytopathic effect were observed by cytopathic effect (CPE), the cytopathic inhibition rate (ER) and therapeutic index (TI).2.2 To study the anti-H1N1 influenza virus effects of the main ingredients in MLQCTo observe the anti-H1N1 influenza virus effects of baicalin, liquiritin, aspartic acid, glutamic acid and leucine from MLQC, the antiviral effect of these ingredients on A549 cells were detected by using MTT. CPE, ER and TI were used to represent the influences of these drugs on antiviral effects.2.3 To optimize the combination of the main active ingredients and analysis the therapeutic basis of MLQCThe combination of the main active ingredients from MLQC was optimized by using uniform design. The main ingredient on antiviral effect, interaction between these ingredients and contribution to antiviral effect were destribed by analyzed by contour plots and analyzed by using Minitab 17.0. According to antiviral effects, the mathematical model and regression equations between the factors were obtained, then to find and validate the best ratio of these active ingredients with the highest anti- H1N1 influenza virus effect.2.4 To study the antiviral molecular mechanisms of MLQCThe antiviral mechanisms of MLQC were predicted via constructing pharmacology network through collecting the chemical ingredients, disease targets and relevant proteins from databases, with these information we can analyze the correlation between ingredients and targets. The immune signal pathways related to H1N1 influenza virus and drugs were found, from which the antiviral mechanism of MLQC was predicted successfully.Results3.1 The antiviral activity of MLQCMLQC had a significant effect on protecting A549 cells from intrusion, when the concentration was 1250μg/mL (Colculated with the content of ME), the antiviral rate was 91.37±1.055%, IC50 was 314.808μg/mL and TI was 20.889. Microemulsion had inhibitory effect on the pathogenic effect of influenza virus to a certain extent, the antiviral rate was 67.573±2.628% with the concentration of 1250μg/mL (Colculated with the content of ME), IC50 was 769.178μg/mL and TI was 9.078.3.2 The antiviral activity of the main ingredients in MLQCThe main ingredinets in MLQC were baicalin, liquirtin, leucine, aspartic acid and glutamic acid in preliminary experiments. The TC50 of baicalin on H1N1 virus was 166.59μg/mL, it had a significant inhibitory effect on the pathogenic effect of influenza virus. When the concentration was 50μg/mL, the antiviral rate was 87.810±1.905%, IC50 and TI was 4.313 and 38.63, respectively. The antiviral rate of liquirtin was 52.959±4.650% when the concentration was 25μg/mL, IC50 was 20.464μg/mL and TI was 4.15. These three amino acids almost had no toxicity to A549 cells at the maximum diluted concentration. They could change the cell morphology and had no inhibitory effect on the pathogenic effect of influenza virus.3.3 The optimized combination and therapeutic basis of MLQCThere was a certain relationship between the five ingredients which had different auxiliary role in antiviral efficiency according to mathematical model and equivalent diagrams. X2 (baicalin), followed by X1 (liquiritin), has greater antiviral effects compared with leucine and aspartic acid, while X5 (glutamic acid) has a certain effect. The regression equation also provided that some interactions were existed between baicalin and liquiritin, aspartic acid and leucine, baicalin, leucine, and aspartic acid. From contour plots we found that with the constant increasing concentration of baicalin, antiviral effect became larger when the concentrations of liquiritin, aspartic acid remained unchanged, leucine showed negative correlation in these plots. The best antiviral effect reached 85.34±4.72% when the optimized concent ratio of liquiritin:baicalin:leucine:aspartic acid:glutamic acid was 0.11:0.40:1.8: 10:20.3.4 The antiviral mechanisms of MLQCIn microemulsion and MLQC network, the chemical ingredients can affect the H1N1 virus targets directly and indirectly. Cholesterol and lecithin in microemulsion can indirectly inhibit influenza virus induced cell lesions through activatiing some Toll like receptors. More than 40 chemical molecules including baicalin, liquiritin, leucine, aspartic acid and glutamic acid, each has a strong interaction with 3 or more targets, maybe occupy some key positions in the antiviral mechanism of MLQC as most of these targets were distributed in viral replication, immune signal transduction and so on.38 main nodes, which were enriched from the compound network, have been participated in the immune response such as toll like singling pathway, JAK-STAT cascade regulation and other 7 signal pathways. Maybe the active ingredients in MLQC can activate TLRs, MAPKs or other immunologic factors by regulating the innate immune response, then promote the secretion of regulatory cytokines and reduce the expression of inflammatory factors in order to inhibit the H1N1 virus replication and transfection in the body.ConclusionThe MLQC played a good role in anti-H1N1 influenza virus, the microemulsion had antiviral effect, and maybe played a supporting role to a certain extent.Baicalin had a significant effect of anti-H1N1 influenza virus in compound Longqincao, and so did liquiritin to a certain extent. But the three amino acids in earthworm could not against H1N1 influenza virus effectively.The combinated ingredients with the best antiviral effect was obtained, in which each of ingredients had a certain contribution, baicalin, followed by liquiritin, had the greatest working for the antiviral activity of MLQC, and glutamate also played a subtle effect. Each ingredient which has an effect on antiviral activity had an interaction with other ingredients. The best anti-H1N1 influenza virus activity of the combinated ingredients revealed that the antiviral effect of MLQC, maybe determined by a variety of ingredients rather than one kind.From the network pharmacology, the antiviral effect of MLQC may be mainly associated with the innate immune regulation such as Toll like signal pathway, JAK-STAT and other pathways, that activate the related immune factors and regulate the final cytokines expression levels to play the role of anti H1N1 influenza virus. |
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