Gansukh Enkhtaivan, Kazibwe Zakayo, Pandurangan Muthuraman, Judy Gopal, Kim Doo Hwan
Department of Bio-resources and Food Science, Konkuk University, Seoul 143-701, South Korea.
Department of Bio-resources and Food Science, Konkuk University, Seoul 143-701, South Korea..
Phytomedicine. 2016 Aug 15;23(9):958-67. doi: 10.1016/j.phymed.2016.06.001. Epub 2016 Jun 3.
Influenza virus is still at large and seriously affects social welfare and health. Dianthus superbus is a well-known medicinal plant widely used in Mongolian and Chinese traditional medicine for anti-inflammatory purposes.
To investigate the influence of this novel herbal medicinal product over virus infection and virus-induced symptoms
Quercetin-7-O-glucoside was isolated by bioassay (anti-influenza)-guided fractionation. The structural elucidation was made with 1H-NMR and 13C-NMR. Influenza A/Vic/3/75 (H3N2), A/PR/8/34 (H1N1), B/Maryland/1/59 and B/Lee/40 viruses were used for the evaluation of the antiviral activity. Virus-induced reactive oxygen species and autophagy formation levels were studied. The antiviral mechanism was elucidated via time-dependent, pre-, post-incubation assay methods. The viral RNA replication inhibition of Q7G was analyzed using quantitative RT-PCR method. The blocking of polymerase basic protein subunits of influenza viral RNA polymerase by Q7G was detected by in silico molecular docking assays using AutoDock Vina program with m(7)GTP. Additionally, Q7G was tested against M-MuLV RNA polymerase.
Q7G was not cytotoxic (CC50>100µg/ml) in MDCK cells and it showed 3.1µg/ml, 6.61µg/ml, 8.19µg/ml and 5.17µg/ml IC50 values against influenza A/PR/8/34, A/Vic/3/75, B/Lee/40 and B/Maryland/1/59 virus strains, respectively. Treatment of Q7G highly reduced ROS and autophagy formation induced by influenza virus infection. Q7G did not reduce NA activity and did not directly interact with the virus particles. Since viral RNA synthesis was blocked by treatment of Q7G. We targeted viral RNA polymerase for further probing. Interestingly, the binding energy of Q7G on viral PB2 protein was -9.1kcal/mol and was higher than m(7)GTP recorded as -7.5kcal/mol. It also was observe to block M-MuLV RNA polymerase.
Isolated compound Q7G showed strong inhibition activity against influenza A and B viruses. It also reduced virus-induced ROS and autophagy formation. Q7G does not directly bind to the virus particles and did not affect NA activity. These results indicated that Q7G inhibits viral RNA polymerase, and that it occupies the binding site of m(7)GTP on viral PB2 protein.
流感病毒仍在肆虐，严重影响社会福利和健康。瞿麦是一种著名的药用植物，在蒙药和中药中广泛用于抗炎。
研究这种新型草药产品对病毒感染及病毒诱导症状的影响。
通过生物测定（抗流感）引导的分级分离法分离出槲皮素-7-O-葡萄糖苷。利用1H-NMR和13C-NMR进行结构解析。使用甲型流感病毒/维多利亚/3/75（H3N2）、甲型流感病毒/PR/8/34（H1N1）、乙型流感病毒/马里兰/1/59和乙型流感病毒/李/40毒株评估抗病毒活性。研究病毒诱导的活性氧和自噬形成水平。通过时间依赖性、预孵育和后孵育测定方法阐明抗病毒机制。使用定量RT-PCR方法分析Q7G对病毒RNA复制的抑制作用。使用AutoDock Vina程序结合m(7)GTP，通过计算机模拟分子对接试验检测Q7G对流感病毒RNA聚合酶基本蛋白亚基的阻断作用。此外，还对Q7G针对莫洛尼鼠白血病病毒（M-MuLV）RNA聚合酶进行了测试。
Q7G在MDCK细胞中无细胞毒性（CC50>100µg/ml），其对甲型流感病毒/PR/8/34、甲型流感病毒/维多利亚/3/75、乙型流感病毒/李/40和乙型流感病毒/马里兰/1/59毒株的IC50值分别为3.1µg/ml、6.61µg/ml、8.19µg/ml和5.17µg/ml。Q7G处理显著降低了流感病毒感染诱导的活性氧和自噬形成。Q7G不降低神经氨酸酶（NA）活性，也不与病毒颗粒直接相互作用。由于Q7G处理阻断了病毒RNA合成，我们将病毒RNA聚合酶作为进一步探究的靶点。有趣的是，Q7G与病毒PB2蛋白的结合能为-9.1kcal/mol，高于记录的m(7)GTP的-7.5kcal/mol。还观察到它能阻断M-MuLV RNA聚合酶。
分离得到的化合物Q7G对甲型和乙型流感病毒显示出强大的抑制活性。它还降低了病毒诱导的活性氧和自噬形成。Q7G不直接与病毒颗粒结合，也不影响NA活性。这些结果表明Q7G抑制病毒RNA聚合酶，并占据了m(7)GTP在病毒PB2蛋白上的结合位点。
