大黃與板藍根抗日本腦炎病毒功效之研究; Study on the antiviral ability of Rheum palmatum and Isatis indigotica against Japanese encephalitis virus

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Title:	大黃與板藍根抗日本腦炎病毒功效之研究;Study on the antiviral ability of Rheum palmatum and Isatis indigotica against Japanese encephalitis virus
Authors:	鄒依芸;Yi-Yun Tsou
Contributors:	中國醫藥大學：藥學系
Keywords:	板藍根;大黃;日本腦炎;Rheum palmatum;Isatis indigotica;Japanese encephalitis virus
Date:	2008-07-23
Issue Date:	2009-08-13 10:53:59 (UTC+8)
Abstract:	板藍根及大黃，在臨床上以證實有許多功效，包括抗細菌、抗病毒、抗腫瘤之效果。本研究的目的在於評估板藍根及大黃之粗抽物及指標成分對於抗日本腦炎病毒功效之探討。首先利用MTT方法分析藥物濃度百分之五十的毒殺效果 ( 50% Cytotoxicity Concentration, CC50)，以及病毒蝕斑試驗來分析板藍根、大黃及其指標成分對日本腦炎病毒之抑制百分之五十之病毒濃度 ( 50% Inhibiting Concentration, IC50)。板藍根乙酸乙酯粗抽物之 IC50為91.57±3.54 μg/mL，板藍根甲醇粗抽物之 IC50為78.47±3.06 μg/mL，板藍根指標成分 indigo之 IC50為37.49±3.18 μg/mL，板藍根指標成分 indirubin之 IC50為13.68±2.83 μg/mL，大黃甲醇粗抽物之 IC50為15.04±5.15 μg/mL，大黃水粗抽物之 IC50為51.41±4.95 μg/mL，大黃指標成分chrysophanol 之 IC50為15.82±2.62 μg/mL，大黃指標成分 aloe-emodin之 IC50為17.39±0.58 μg/mL。發現板藍根粗萃物、大黃萃取物及其指標成分對於三株細胞分別有不同的毒性，而在利用抑制日本腦炎病毒之 IC50來決定接下來實驗所需的藥物。以 virucidal effect 實驗來抑制日本腦炎病毒，發現板藍根乙酸乙酯、板藍根甲醇濃度 100 μg/mL、10 μg/mL、1 μg/mL抑制日本腦炎病毒分別為 54.58%、32.49%、26.35%， 62.90%、45.26%、22.42%。於濃度 10 μg/mL、1 μg/mL、0.1 μg/mL抑制日本腦炎病毒分別為61.94%、35.48%、27.81%， 78.71%、62.24%、30.65%。之後以即時定量 PCR（Real-time PCR）實驗顯示出，在細胞以藥物及病毒處理後，會造成日本腦炎病毒複製倍數下降，顯示出藥物可以抑制日本腦炎病毒動態複製情形。。另一方面以日本腦炎病毒 NS2B-NS3蛋白脢活性分析，發現 indigo 及 indirubin 有抑制日本腦炎病毒 NS2B-NS3蛋白切割能力。進一步利用流式細胞儀來測試細胞 NO 產生，結果顯示板藍根、大黃及其指標成分皆會導致 NO 量的增加。進一步利用 Fluorescent ISRE-SEAP 報導基因分析證實板藍根及指標成分會促使細胞產生干擾素之抗病毒作用。 Isatis indigotica and Rheum palmatum have been demonstrated to have various clincal activities, including antibacterial, antiviral and anti-tumor effects. The goal of this study is to investigate the antiviral effects of Isatis indigotica, Rheum palmatum and their marker compounds against Japanese encephalitis virus ( JEV) in vitro cell culture assay. First, MTT assay was used measured for 50% cytotoxicity concentration (CC50) of each extract and marker compounds. The plaque assay was carried out fore detecting 50% inhibiting concentration ( IC50) of Isatis indigotica, Rheum palmatum and their marker compounds to against JEV. The IC50 values of the ethyl acetate and methanol extract of Isatis indigotica against JEV were 91.57±3.54 μg/mL and 78.47±3.06 μg/mL , respectively. The IC50 values of the indigo and indirubin against JEV were 37.49±3.18 μg/mL and 13.68±2.83 μg/mL, respectively. Methanol and water extracts of Rheum palmatum against JEV were the IC50 of 15.04±5.15 μg/mL and the IC50 of 51.41±4.95 μg/mL , respectively. Chrysophanol and aloe-emodin against JEV was the IC50 of 15.82±2.62 μg/mL and the IC50 of 17.39±0.58 μg/mL, respectively. Testing for virucidal effect of ethyl acetate extract of Isatis indigotica ( 100 μg/mL , 10 μg/mL , 1 μg/mL ) inhibit on JEV was 54.58%、32.49%、26.35%, methanol of extract of Isatis indigotica ( 100 μg/mL , 10 μg/mL , 1 μg/mL ) inhibit on JEV was 62.90%、45.26%、22.42%. Indigo and indirubin( 10 μg/mL、1 μg/mL、0.1 μg/mL ) inhibit on JEV was 1 61.94%、35.48%、27.81% and 78.71%、62.24%、30.65%, respectively.Isatis indigotica and Rheum palmatum have been demonstrated to have various clincal activities, including antibacterial, antiviral and anti-tumor effects. The goal of this study is to investigate the antiviral effects of Isatis indigotica, Rheum palmatum and their marker compounds against Japanese encephalitis virus ( JEV) in vitro cell culture assay. First, MTT assay was used measured for 50% cytotoxicity concentration (CC50) of each extract and marker compounds. The plaque assay was carried out fore detecting 50% inhibiting concentration ( IC50) of Isatis indigotica, Rheum palmatum and their marker compounds to against JEV. The IC50 values of the ethyl acetate and methanol extract of Isatis indigotica against JEV were 91.57±3.54 μg/mL and 78.47±3.06 μg/mL , respectively. The IC50 values of the indigo and indirubin against JEV were 37.49±3.18 μg/mL and 13.68±2.83 μg/mL, respectively. Methanol and water extracts of Rheum palmatum against JEV were the IC50 of 15.04±5.15 μg/mL and the IC50 of 51.41±4.95 μg/mL , respectively. Chrysophanol and aloe-emodin against JEV was the IC50 of 15.82±2.62 μg/mL and the IC50 of 17.39±0.58 μg/mL, respectively. Testing for virucidal effect of ethyl acetate extract of Isatis indigotica ( 100 μg/mL , 10 μg/mL , 1 μg/mL ) inhibit on JEV was 54.58%、32.49%、26.35%, methanol of extract of Isatis indigotica ( 100 μg/mL , 10 μg/mL , 1 μg/mL ) inhibit on JEV was 62.90%、45.26%、22.42%. Indigo and indirubin( 10 μg/mL、1 μg/mL、0.1 μg/mL ) inhibit on JEV was 1 61.94%、35.48%、27.81% and 78.71%、62.24%、30.65%, respectively.Moreover, real-time PCR demonstrated the inhibition of JEV titers by Isatis indigotica, Rheum palmatum and their marker compounds.Otherwise, Isatis indigotica and its marker compounds inhibited the proteolytic activity of the JEV NS2B-NS3 protease. Furthermore, flow cytometry assay showed that Isatis indigotica, Rheum palmatum and their marker compounds stimulated the NO production in human promonocyte cells. Moreover, interferon stimulated response element- SEAP (ISRE-SEAP) cis-acting reporter assay indicated that Isatis indigotica and its marker compounds induced the cis-acting reporter activity, being responsible for the interferon-mediated antiviral ability.
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