葛根湯及其相關桂枝成分抗腸病毒71型活性及其作用機轉研究

Anti-enterovirus 71 activity and mechanism of Ge-Gen-Tang and its related pure compounds of Cinnamomum cassia

尹照雯(Jau-Wen Ying)

指導教授 ： 蔣連財

高雄醫學大學/醫學院/醫學研究所/碩士(2009年)

摘要

中文摘要
腸病毒年年肆虐，腸病毒71型(EV71)，沒有臨床用藥和疫苗，因此本論文研究中草藥來防治EV71感染之可行性。
葛根湯曾報導有抗流行性感冒、疱疹病毒和腸病毒71型感染，因此選擇了葛根湯和桂枝，利用XTT分析法，研究抗病毒(EV71)之活性成分和機轉，結果顯示：葛根湯與桂枝抑制病毒感染50%的劑量(IC50)分別為0.93 ± 0.01 μg/ml和1.70 ± 0.21 μg/ml，選擇指數(SI)分別是大於3000和294，証實這兩種藥物都可以有效地(IC50低)以及安全地(SI高)抑制EV71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。
桂枝相關純化合物trans-cinnamic acid (TCA)、cinnamaldehyde (Cin)、cinnamayl acetate (CA)、benzaldehyde (Ben)、cuminaldehyde (Cum)、vanillin (Van)、2-methoxycinnamic acid (2-Meth-acid)、2-methoxycinnamaldehyde (2-Meth-ald)的抗病毒實驗顯示：TCA的IC50為34.36 ± 0.89 μM，SI值為39.35；Cin的IC50為1.57 ± 0.05 μM，SI值為99.08；CA的IC50為27.73 ± 3.14 μM，SI值為34.07；Ben的IC50為0.21 ± 0.01 μM，SI值為13128.57；Cum的IC50為0.89 ± 0.04 μM，SI值為1245.88；Van的IC50為1.65 ± 0.39 μM，SI值為1575.50；2-Meth-acid的IC50為10.06 ± 2.01 μM，SI值為94.58；2-Meth-ald的IC50為0.94 ± 0.15 μM，SI值為162.14，顯示benzaldehyde、cuminaldehyde、cinnamaldehyde、vanillin以及2-methoxycinnamaldehyde都有很好的抗病毒效果，而且皆是新發現。
時間效應顯示benzaldehyde、cuminaldehyde和cinnamaldehyde在感染前後皆有很好的抗病毒活性，因此推論這三種純化合物可能是桂枝的預防和治療EV71感染的重要成分。
藥物預防EV71感染的作用機轉結果顯示：benzaldehyde抑制病毒附著之IC50值為0.55 ± 0.39 μM；抑制病毒穿透能力之IC50值為0.07 ± 0.01 μM~0.21 ± 0.03 μM。
藥物治療EV71感染的作用機轉結果顯示：benzaldehyde抑制感染後病毒複製並不是經由刺激細胞產生干擾素。
本論文結果顯示葛根湯及桂枝，都有預防和治療EV71感染的潛力，未來可以再深入研究其抗病毒的作用機轉，而本研究結果也可以提供未來研發抗腸病毒71型藥物的參考。

關鍵字

腸病毒71型 ； 葛根湯 ； 桂枝

並列摘要

Abstract
Enterovirus 71 (EV71) is endemic spread all of the year in the world. There is still no effective agent and vaccine could be used. In this study, we wanted to seek potential herbal candidate to prevent EV71 infection.
Ge-Gen-Tang (GGT) is a traditional Chinese herbal remedy and commonly used to treat infectious disease including common cold. GGT has been found to be active against influenza virus, herpesvirus type 1, and EV71. Therefore, we tested the GGT and its related ingredients, Cinnamomum cassia Blume against EV71 by XTT reduction assay. The results showed that IC50 of the crude extracts of GGT and C. cassia against EV71 infection were 0.93 ± 0.01 μg/ml and 1.70 ± 0.21 μg/ml, respectively. The selectivity index (SI: CC50/IC50) of GGT and C. cassia were larger than 3000 and 294, respectively. These results indicated that GGT and C. cassia were effective (low IC50 value) and safe (high SI value) against EV71.
The result of time course assay indicated that GGT and C. cassia exhibited their anti-EV71 activity both before and after EV71 infection. Therefore, they had both preventive and therapeutic potential.
Later, we proved the effective pure pompounds of C. cassia to be trans-cinnamic acid, cinnamaldehyde, cinnamyl acetate, benzaldehyde, cuminaldehyde, vanillin, 2-methoxycinnamic acid, and 2-methoxycinnamaldehyde. Their IC50 against EV71 of benzaldehyde, cuminaldehyde, 2-methoxycinnamaldehyde, cinnamaldehyde, vanillin, 2-methoxycinnamic acid, cinnamyl acetate, and trans-cinnamic acid against EV71 infection were 0.21 ± 0.01 μM, 0.89 ± 0.04 μM, 0.94 ± 0.15 μM, 1.57 ± 0.05 μM, 1.65 ± 0.39 μM, 10.06 ± 2.01 μM, 27.73 ± 4.14 μM, and 34.36 ± 0.89 μM, respectively.
The selectivity indexes (SI: CC50/IC50) of benzaldehyde, vanillin, cuminaldehyde, 2-methoxycinnamaldehyde, cinnamaldehyde, 2-methoxycinnamic acid, trans-cinnamic acid, and cinnamyl acetate were 13128.57, 1575.50, 1245.88, 162.14, 99.08, 94.58, 39.35, and 34.07 respectively. Our results revealed several novel anti-EV71 agents, such as benzaldehyde, cuminaldehyde, cinnamaldehyde, vanillin, and 2-methoxycinnamaldehyde.
In order to understand the anti-EV71 mechanisms, we had time course assay, attachment assay, and interferon assay. The results of time course assay showed that benzaldehyde, cuminaldehyde, and cinnamaldehyde exhibited their anti-EV71 activity before and after EV71 infection. Therefore, all of these agents had preventive and therapeutic potential. The results of attachment assay and penetration assay of Benzaldehyde showed that the IC50 against EV71 attachment activity was 0.55 ± 0.39 μM. Its IC50 against EV71 penetration activity after 10min~60min treatment ranged from 0.07 ± 0.01 μM to 0.21 ± 0.03 μM, respectively.
Benzaldehyde did not stimulate interferon production. Therefore, its anti-EV71 activity was not mediated by interferon.
Our results have proven that GGT, C. cassia and several its pure compounds are effective to manage EV71 infection by inhibiting EV71 induced cytotoxicity, by inhibiting viral attachment and/or penetration. Our results could be helpful to develop effective anti-EV71 agents.

並列關鍵字

enterovirus 71 ； Ge-Gen-Tang ； Cinnamomum cassia

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