【摘要】：板藍根是十字花科植物菘藍的干燥根,具有清熱解毒,涼血利咽之功效,臨床多用于治療各種病毒感染引起的疾病。目前,對于板藍根抗病毒藥效物質(zhì)基礎(chǔ)及作用機制研究有了一定進展,但仍沒有發(fā)現(xiàn)確切的抗病毒藥效成分。研究中的問題集中體現(xiàn)在:(1)體外研究較多,體內(nèi)研究較少。(2)化學(xué)成分研究較多,但抗病毒相關(guān)活性篩選不夠全面。(3)單純抗病毒研究較多,但整體調(diào)控作用研究較少。本課題針對以上三個問題展開相關(guān)研究,為板藍根藥效物質(zhì)基礎(chǔ)的發(fā)現(xiàn)和抗病毒藥理作用機制的闡明奠定基礎(chǔ)。(1)板藍根血清藥物化學(xué)研究。利用HPLC法對血漿樣品的前處理和色譜條件進行了全面考察,建立了板藍根水提液含藥血漿的指紋圖譜,板藍根水提液含藥血漿樣品與空白血漿樣品圖譜對照,初步確定了板藍根灌胃給藥后的含藥血漿中主要含有10個移行成分。采用LC-MS技術(shù)分析了板藍根水提液、空白血漿、含藥血漿中的化學(xué)成分,最終在板藍根水提液中鑒定出59個化學(xué)成分;含藥血漿圖譜以空白血漿圖譜為背景進行扣除背景處理,并以板藍根水提液中檢測到的59個化學(xué)成分為數(shù)據(jù)庫,運用XIC Manager功能對扣除背景后的含藥血漿圖譜進行蹄查和鑒定,發(fā)現(xiàn)了20種入血成分,根據(jù)二級質(zhì)譜裂解規(guī)律和相關(guān)文獻報道,鑒定出了其中12個化學(xué)成分,并對4(3H)喹唑酮、色胺酮、異牡荊素、表告依春4個成分同標準品進行比對,進一步確認了化學(xué)結(jié)構(gòu)。(2)板藍根潛在藥效成分抑制神經(jīng)氨酸酶(NA)、血凝素(HA)的直接抗病毒作用研究。利用計算機分子對接技術(shù)對已從板藍根分離得到的化學(xué)成分進行了全面篩選,得到28個對神經(jīng)氨酸酶可能有抑制活性的化學(xué)成分,3個對血凝素有抑制作用的化學(xué)成分,其中依靛藍酮、3-(2'羥苯基)-4(3H)-喹唑酮、3-(2'羧基苯基)-4(3H)-喹唑酮、色胺酮在板藍根水提液灌胃后的大鼠血漿中能夠檢測到。采用表面等離子體共振(SPR)技術(shù),成功篩選到了 13種化合物與NA蛋白有相互作用;篩選到靛藍與HA蛋白有相互作用,并得到了其相互作用的動力學(xué)及親和力參數(shù)。采用以4-MUNANA為底物的熒光法,對前期通過分子對接和SPR篩選出的板藍根各潛在藥效成分,進行了抑制流感病毒神經(jīng)氨酸酶的活性驗證,檢測結(jié)果表明,4(3H)-喹唑酮和甘草素具有較好的抑制活性,(+)-丁香樹脂醇、大黃素、靛藍、水楊酸、脫落酸、β-谷甾醇、3-醛基吲哚、吲哚-3-乙腈具有較弱的抑制活性;腺嘌呤、棕櫚酸、對羥基苯甲酸無抑制活性;通過紅細胞凝集實驗證實了靛藍具有抑制HA活性的作用。(3)板藍根潛在藥效成分對維甲酸誘導(dǎo)基因蛋白Ⅰ(RIG-Ⅰ)和Toll樣受體3(TLR3)介導(dǎo)的天然免疫信號通路調(diào)控作用的研究。通過PCR、ELISA、Western Blot等檢測方法研究了板藍根含藥血漿對RSV/ploy(I:C)感染后RAW264.7細胞中RIG-Ⅰ和TLR3介導(dǎo)的抗病毒天然免疫信號通路關(guān)鍵信號分子的影響,結(jié)果表明:當(dāng)細胞被病毒感染時,細胞中RIG-Ⅰ和TLR3信號通路關(guān)鍵信號分子mRNA轉(zhuǎn)錄水平和蛋白表達水平都發(fā)生上調(diào),從而導(dǎo)致IFN-β的高表達。板藍根的抗病毒機制可能是通過下調(diào)病毒感染所誘導(dǎo)的RIG-Ⅰ和TLR3信號通路關(guān)鍵信號分子的高表達,從而下調(diào)IRF3的磷酸化,最終適度抑制病毒感染誘導(dǎo)的IFN-β的高表達,使IFN-β的表達量維持在一個平衡點,既能起到有效的抗病毒作用,又能減輕炎癥和組織損傷。
[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，

本文編號：2268179