葛根湯及其相關甘草成份抗腸病毒71型活性及其作用機轉之研究

Anti-enterovirus 71 activity and mechanism of Ge-Gen-Tang and its related pure ingredients of Glycyrrhiza glabra

霍宣瑋(Shuan-wei Huo)

指導教授 ： 蔣連財

高雄醫學大學/醫學院/醫學研究所/碩士(2010年)

摘要

1998年，台灣爆發腸病毒大流行，造成恐慌，腸病毒的嚴重性也開始被重視，台灣地區位在亞熱帶，所以全年都有感染病例出現，仍以夏季為主要流行季節。腸病毒感染併發重症之型別以腸病毒71型 ( EV 71 ) 為主，目前為止是沒有臨床用藥和疫苗，因此本論文目的在研究中草藥來防治腸病毒71型 ( EV 71) 感染之可行性。
葛根湯曾報導可以抑制流行性感冒病毒、單純疱疹病毒、和腸病毒71型感染，因此選擇了葛根湯和其單方【甘草】，利用細胞病變抑制法( cytopathic reduction assay ) 和病毒溶斑抑制法 ( plaque reduction assay )，研究抗病毒 EV 71 之活性成份和機轉，結果顯示：葛根湯與甘草抑制病毒感染50%的劑量( IC50 )分別為1.82 ± 0.46 μg/ml和1.36 ± 0.19 μg/ml，選擇指數( SI )分別是大於1648和2206，証實這兩種藥物都可以有效 ( IC50值低 ) 以及安全 ( SI值高) 抑制EV 71感染。時間效應實驗發現感染前後皆有抗病毒活性，顯示皆有預防和治療之功效。
甘草相關純化合物抗病毒實驗顯示皆有很好的抗EV 71效果： (1) 三萜類化合物: glycyrrhizin的IC50為1.30 ± 0.07 μM，SI值為215.3；18 α - glycyrrhetinic acid的IC50為1.61 ± 0.06 μM，SI值為117.5；18 β - glycyrrhetinic acid的IC50為1.56 ± 0.04 μM，SI值為109.5；(2) 類黃酮化合物 : liquiritin的IC50為2.01 ± 0.35 μM，SI值為61.6；isoliquiritigenin的IC50為1.80 ± 0.07 μM ， SI值為94；kaempferol的IC50為1.53 ± 0.11 μM，SI值為125.7；(3) 異黃酮化合物: glabridin的IC50為2.16 ± 0.31 μM，SI值為75.7；genistein的IC50為 1.60 ± 0.19 μM，SI值為103.9。
時間效應顯示甘草成份中的liquiritin、isoliquiritigenin和glabridin在感染前後皆有很好的抗病毒活性，而且其預防病毒EV 71吸附作用和病毒穿透細胞能力均會隨著劑量上升而增加。
干擾素分泌量結果顯示甘草成份中的liquiritin、isoliquiritigenin 和 glabridin能有效誘生干擾素，但是均無法有效促進已經感染病毒的細胞分泌干擾素，因此抑制感染後病毒複製的機轉不是經由干擾素。
依據甘草成份實際含量之抗腸病毒71型活性分析顯示：liquiritin、isoliquiritigenin和glabridin三種甘草純化合物混合後抑制EV 71感染活性為80.9 ± 2.8 %，其抗EV 71活性和甘草的93.4 ± 1.4 % 接近，因此liquiritin、isoliquiritigenin和glabridin可能是甘草抗EV 71活性的主要成份。
抑制病毒溶斑實驗結果進一步證實葛根湯、甘草和甘草成份中的glycyrrhizin、liquiritin、isoliquiritigenin和 glabridin均可以有效抑制EV 71病毒 ，而且呈現劑量效應。
本論文研究結果顯示葛根湯、甘草和八種甘草純化合物，皆有抑制腸病毒71型感染活性，甘草抗病毒主要成分可能是liquiritin、isoliquiritigenin和glabridin，未來可以再深入探討其抑制感染後EV 71病毒複製以及藥物應用性。

關鍵字

葛根湯 ； 甘草 ； 腸病毒71型

並列摘要

In 1998, the endemic enterovirus was widely spread over many regions in Taiwan. After the virus caused the public to panic, the Taiwanese government began to aware of the severity of this intestinal virus. Taiwan is a subtropical country ; therefore the infection of endemic enterovirus is usually recurrent and circulate throughout the year, especially in the summer. The serious complications of enterovirus infections are typically caused by enterovirus 71 ( EV 71 ) infection which so far does not have any effective drug, nor vaccine. Therefore, the purpose of this thesis was to find the feasibility and the impact of Chinese herbal medicine on EV 71 infection.
Ge-Gen-Tang 【GGT】 is a traditional Chinese medicine, which has been reported to inhibit Influenza virus, Herpesvirus, and EV 71 infection. Therefore, the GGT and its ingredient, licorice 【Glycyrrhiza glabra】, were chosen to study their anti-EV 71 activity by cytopathic reduction assay and plaque reduction assay. The results showed that IC50 of the crude extracts of GGT and G. glabra ( GG ) against EV 71 infection were 1.82 ± 0.46 μg/ml and 1.36 ± 0.19 μg/ml, respectively. The selectivity index of GGT and GG were larger than 1648 and 2206, respectively. These results indicated that GGT and G. glabra were effective ( low IC50 value ) and safe ( high SI value ) . The data of time course assay indicated that GGT and GG were effective both before and after EV 71 infection. Therefore, GGT and GG had preventive and therapeutic potentials.
Later, pure ingredients of GG, such as glycyrrhizin, 18 α - glycyrrhetinic acid, 18 β – glycyrrhetinic acid, liquiritin, isoliquiritigenin, kaempferol , glabridin and genistein, were proved to be effective. The IC50 of glycyrrhizin, kaempferol, 18 β – glycyrrhetinic acid, genistein, 18 α - glycyrrhetinic acid , isoliquiritigenin, liquiritin and glabridin against EV 71 infection were 1.30 ± 0.07 μM , 1.53 ± 0.11 μM, 1.56 ± 0.04μM , 1.60 ± 0.19 μM , 1.61 ± 0.06 μM, 1.80 ± 0.07 μM, 2.01 ± 0.35 μM and 2.16 ± 0.31 μM, respectively. The selectivity indexes ( SI: CC50 / IC50 ) of glycyrrhizin, kaempferol, 18α- glycyrrhetinic acid, 18β– glycyrrhetinic acid, genistein, isoliquiritigenin, glabridin and liquiritin were 215.3, 125.7, 117.5, 109.5, 103.9, 94, 75.7 and 61.6, respectively.
In order to understand their anti-EV 71 mechanisms, time course assay, attachment assay, penetration assay and interferon ELISA assay were done. The time course assay showed that liquiritin, isoliquiritigenin and glabridin have excellent antiviral activities during the pre-infection and post-infection periods. The results of the attachment and penetration assays against the EV 71 infection suggest that the ingredients of G. glabra, namely liquiritin, isoliquiritigenin and glabridin, exhibited excellent anti-EV 71 activities with a dose - dependent fashion .
Liquiritin, isoliquiritigenin and glabridin stimulated interferon secretion only before viral infection. Therefore, the mechanism of anti-EV 71 activity after the virus infection was not mediated by interferon.
The combination of liquiritin, isoliquiritigenin and glabridin shows its anti-EV 71 activity of 80.9 ± 2.8 % , which was similar with that of GG ( 93.4 ± 1.4 % ) . This finding might imply that they are the main active ingredients of G. glabra.
The plaque reduction assays confirmed the dose-dependent effect of GGT , GG and the ingredients of GG , namely glycyrrhizin, liquiritin, isoliquiritigenin and glabridin against EV 71 infection .
My results demonstrated that GGT, GG and the eight pure ingredients of licorice (GG) , especially liquiritin, isoliquiritigenin and glabridin are effective to suppress the EV 71 infection.
Further elucidation of the mechanism to inhibit EV 71 replication after infection might be helpful to develop effective anti-EV 71 agents.

並列關鍵字

Ge-Gen-Tang ； Glycyrrhiza glabra ； enterovirus 71

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