板蓝根潜在药效成分的发现及其作用机制的研究
发布时间：2018-10-13 09:54
【摘要】：板蓝根是十字花科植物菘蓝的干燥根,具有清热解毒,凉血利咽之功效,临床多用于治疗各种病毒感染引起的疾病。目前,对于板蓝根抗病毒药效物质基础及作用机制研究有了一定进展,但仍没有发现确切的抗病毒药效成分。研究中的问题集中体现在:(1)体外研究较多,体内研究较少。(2)化学成分研究较多,但抗病毒相关活性筛选不够全面。(3)单纯抗病毒研究较多,但整体调控作用研究较少。本课题针对以上三个问题展开相关研究,为板蓝根药效物质基础的发现和抗病毒药理作用机制的阐明奠定基础。(1)板蓝根血清药物化学研究。利用HPLC法对血浆样品的前处理和色谱条件进行了全面考察,建立了板蓝根水提液含药血浆的指纹图谱,板蓝根水提液含药血浆样品与空白血浆样品图谱对照,初步确定了板蓝根灌胃给药后的含药血浆中主要含有10个移行成分。采用LC-MS技术分析了板蓝根水提液、空白血浆、含药血浆中的化学成分,最终在板蓝根水提液中鉴定出59个化学成分;含药血浆图谱以空白血浆图谱为背景进行扣除背景处理,并以板蓝根水提液中检测到的59个化学成分为数据库,运用XIC Manager功能对扣除背景后的含药血浆图谱进行蹄查和鉴定,发现了20种入血成分,根据二级质谱裂解规律和相关文献报道,鉴定出了其中12个化学成分,并对4(3H)喹唑酮、色胺酮、异牡荆素、表告依春4个成分同标准品进行比对,进一步确认了化学结构。(2)板蓝根潜在药效成分抑制神经氨酸酶(NA)、血凝素(HA)的直接抗病毒作用研究。利用计算机分子对接技术对已从板蓝根分离得到的化学成分进行了全面筛选,得到28个对神经氨酸酶可能有抑制活性的化学成分,3个对血凝素有抑制作用的化学成分,其中依靛蓝酮、3-(2'羟苯基)-4(3H)-喹唑酮、3-(2'羧基苯基)-4(3H)-喹唑酮、色胺酮在板蓝根水提液灌胃后的大鼠血浆中能够检测到。采用表面等离子体共振(SPR)技术,成功筛选到了 13种化合物与NA蛋白有相互作用;筛选到靛蓝与HA蛋白有相互作用,并得到了其相互作用的动力学及亲和力参数。采用以4-MUNANA为底物的荧光法,对前期通过分子对接和SPR筛选出的板蓝根各潜在药效成分,进行了抑制流感病毒神经氨酸酶的活性验证,检测结果表明,4(3H)-喹唑酮和甘草素具有较好的抑制活性,(+)-丁香树脂醇、大黄素、靛蓝、水杨酸、脱落酸、β-谷甾醇、3-醛基吲哚、吲哚-3-乙腈具有较弱的抑制活性;腺嘌呤、棕榈酸、对羟基苯甲酸无抑制活性;通过红细胞凝集实验证实了靛蓝具有抑制HA活性的作用。(3)板蓝根潜在药效成分对维甲酸诱导基因蛋白Ⅰ(RIG-Ⅰ)和Toll样受体3(TLR3)介导的天然免疫信号通路调控作用的研究。通过PCR、ELISA、Western Blot等检测方法研究了板蓝根含药血浆对RSV/ploy(I:C)感染后RAW264.7细胞中RIG-Ⅰ和TLR3介导的抗病毒天然免疫信号通路关键信号分子的影响,结果表明:当细胞被病毒感染时,细胞中RIG-Ⅰ和TLR3信号通路关键信号分子mRNA转录水平和蛋白表达水平都发生上调,从而导致IFN-β的高表达。板蓝根的抗病毒机制可能是通过下调病毒感染所诱导的RIG-Ⅰ和TLR3信号通路关键信号分子的高表达,从而下调IRF3的磷酸化,最终适度抑制病毒感染诱导的IFN-β的高表达,使IFN-β的表达量维持在一个平衡点,既能起到有效的抗病毒作用,又能减轻炎症和组织损伤。
[Abstract]:It has effects in clearing away heat and toxic materials, cooling blood and relieving sore throat, and can be used for treating diseases caused by various viral infections. At present, some progress has been made on the research of the material base and mechanism of the antiviral drug effect of Isatis root, but there is still no exact antiviral efficacy component. The problems in the study are as follows: (1) In vitro studies, there are fewer in vivo studies. (2) The research of chemical composition was much higher, but the screening of anti-virus-related activity was not comprehensive. (3) Only anti-virus research was more, but the overall control effect was less. In order to lay the foundation for the discovery of the drug substance base and the elucidation of the mechanism of antiviral pharmacological action. (1) Chemical study on serum drugs of Isatis indigotica. The pretreatment and chromatographic conditions of plasma samples were comprehensively investigated by HPLC, and the fingerprint of drug-containing plasma was established. It was preliminarily determined that the drug-containing plasma contained 10 transitional components in the drug-containing plasma after oral administration of Isatis root. According to LC-MS technique, the chemical constituents of aqueous extract, blank plasma and drug-containing plasma were analyzed, and 59 chemical components were identified in the aqueous extract of Isatis indigotica. The plasma map of drug-containing plasma was treated with blank plasma map as background. In this paper, 59 chemical components detected in the aqueous extract of Isatis root were used as the database, and the blood-containing plasma map after deducting background was tested and identified by the function of XIC Manager, and 20 kinds of blood-in components were found. According to the law of the second-order mass spectrometry and related literature, 12 chemical components were identified, and 4 (3H) oxycodone, oxycodone, isovitexin were identified, and the comparison of the four components with the standard was given in the table, and the chemical structure was further confirmed. (2) The study on the direct antiviral effects of the potential drug effect components of Isatis indigofera in the inhibition of neuritinase (NA) and haemagglutinin (HA). The chemical components obtained from the separation of the roots were screened by means of the computer molecular docking technique, and 28 neuropeptides which could inhibit the activity were obtained. Three chemical components with inhibitory effect on the hemagglutinin were obtained, including the indigo ketone, 3-(2). 'hydroxyphenyl) -4 (3H)-benzidine, 3-(2') Poly (phenyl) -4 (3H)-bupropion and oxycodone can be detected in rat plasma after oral gavage administration. The interaction of 13 compounds with NA protein was successfully screened by surface plasmon resonance (SPR) technique, and the interaction between indigo and HA protein was selected, and the interaction kinetics and affinity parameters were obtained. Using 4-MUNANA as the substrate, the active verification of the neuramininase of the influenza virus was carried out on the potential drug effect components of Isatis indigenum by molecular docking and SPR screening, and the results showed that the 4 (3H)-dinitrofluoroketone and licorice had better inhibitory activity. (+)-Syringa resin alcohol, Emodin, indigo, salicylic acid, disodium hydrogen phosphate, catechol-oryzene, 3-aldehyde-based disodium hydrogen phosphate, quercetin-3-acetonitrile have a weak inhibitory activity, and the tannin and palmitic acid have no inhibitory activity to hydroxyl benzoic acid; The effect of indigo on HA activity was confirmed by red blood cell agglutination test. (3) The research on the regulation of the natural immune signal pathway mediated by retinoic acid-induced gene protein 鈪，
本文编号：2268179
[Abstract]:It has effects in clearing away heat and toxic materials, cooling blood and relieving sore throat, and can be used for treating diseases caused by various viral infections. At present, some progress has been made on the research of the material base and mechanism of the antiviral drug effect of Isatis root, but there is still no exact antiviral efficacy component. The problems in the study are as follows: (1) In vitro studies, there are fewer in vivo studies. (2) The research of chemical composition was much higher, but the screening of anti-virus-related activity was not comprehensive. (3) Only anti-virus research was more, but the overall control effect was less. In order to lay the foundation for the discovery of the drug substance base and the elucidation of the mechanism of antiviral pharmacological action. (1) Chemical study on serum drugs of Isatis indigotica. The pretreatment and chromatographic conditions of plasma samples were comprehensively investigated by HPLC, and the fingerprint of drug-containing plasma was established. It was preliminarily determined that the drug-containing plasma contained 10 transitional components in the drug-containing plasma after oral administration of Isatis root. According to LC-MS technique, the chemical constituents of aqueous extract, blank plasma and drug-containing plasma were analyzed, and 59 chemical components were identified in the aqueous extract of Isatis indigotica. The plasma map of drug-containing plasma was treated with blank plasma map as background. In this paper, 59 chemical components detected in the aqueous extract of Isatis root were used as the database, and the blood-containing plasma map after deducting background was tested and identified by the function of XIC Manager, and 20 kinds of blood-in components were found. According to the law of the second-order mass spectrometry and related literature, 12 chemical components were identified, and 4 (3H) oxycodone, oxycodone, isovitexin were identified, and the comparison of the four components with the standard was given in the table, and the chemical structure was further confirmed. (2) The study on the direct antiviral effects of the potential drug effect components of Isatis indigofera in the inhibition of neuritinase (NA) and haemagglutinin (HA). The chemical components obtained from the separation of the roots were screened by means of the computer molecular docking technique, and 28 neuropeptides which could inhibit the activity were obtained. Three chemical components with inhibitory effect on the hemagglutinin were obtained, including the indigo ketone, 3-(2). 'hydroxyphenyl) -4 (3H)-benzidine, 3-(2') Poly (phenyl) -4 (3H)-bupropion and oxycodone can be detected in rat plasma after oral gavage administration. The interaction of 13 compounds with NA protein was successfully screened by surface plasmon resonance (SPR) technique, and the interaction between indigo and HA protein was selected, and the interaction kinetics and affinity parameters were obtained. Using 4-MUNANA as the substrate, the active verification of the neuramininase of the influenza virus was carried out on the potential drug effect components of Isatis indigenum by molecular docking and SPR screening, and the results showed that the 4 (3H)-dinitrofluoroketone and licorice had better inhibitory activity. (+)-Syringa resin alcohol, Emodin, indigo, salicylic acid, disodium hydrogen phosphate, catechol-oryzene, 3-aldehyde-based disodium hydrogen phosphate, quercetin-3-acetonitrile have a weak inhibitory activity, and the tannin and palmitic acid have no inhibitory activity to hydroxyl benzoic acid; The effect of indigo on HA activity was confirmed by red blood cell agglutination test. (3) The research on the regulation of the natural immune signal pathway mediated by retinoic acid-induced gene protein 鈪，
本文编号：2268179
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