- 學位論文
Chebulagic acid和Punicalagin對克沙奇病毒A16之抗病毒活性研究
Antiviral Activities of Chebulagic acid and Punicalagin against Human Coxsackievirus A16
摘要
克沙奇病毒A16 (Coxsackievirus A16)隸屬於微小核糖核酸病毒科(Picornaviridae)腸病毒屬(Enterovirus)的成員之一，好發疱疹性咽峽炎(herpangina)及手口足症(hand-foot-mouth disease)，也可能造成嚴重的中樞神經系統受損症狀，甚至死亡。臺灣兒童每年秋夏天面對多種嚴重的腸病毒之威脅，除了腸病毒71型外，克沙奇病毒A16亦為常見的腸病毒類型，而目前臨床上尚無有效的臨床治療藥物及預防疫苗，因此，研發抗病毒潛力藥物有其必要性及迫切性。本研究先以細胞存活率試驗(XTT assay)，利用Rhabdomyosarcoma (RD)細胞與多種天然物成分，嘗試找出試驗藥物之最大耐受濃度，並據此探討天然物成分對克沙奇病毒A16之抗病毒作用，其中發現鞣質類化合物Chebulagic acid (CHLA)和Punicalagin (PUG)具有高度抑制病毒效果。由實驗結果發現，CHLA和PUG之CC50分別為45.62 ± 9.58和76.96 ± 12.07 µM，而在抑制克沙奇病毒A16試驗中，CHLA和PUG呈現抗病毒效力之劑量依賴性現象，且其EC50分別為3.84 ± 0.91和4.61 ± 1.37 µM。而在探討抗病毒機制中，針對病毒生活週期，本研究以先投予藥物(pre-treatment model)、藥物和病毒同時給予(co-addition model)，及感染後再投藥(post-infection model)三種模式分別探討試驗藥物之抗病毒作用機制是否為干擾病毒感染、複製/轉譯或釋出階段，再藉由流式細胞儀偵測病毒附著細胞的表現，並以西方墨點法測定病毒蛋白的含量。結果發現CHLA和PUG 在co-addition模式有顯著的抑制效果，據此得知CHLA和PUG具備抑制克沙奇病毒A16感染階段的潛力，作用機制推斷為干擾病毒感染和附著，而在去病毒活性試驗中，也能發現CHLA和PUG有顯著的效用，而其詳細的抗病毒作用機制尚需要後續更進一步之研究，相信後續研究將有助於釐清該藥物的性質，期望未來可開發成治療克沙奇病毒A16的潛力藥物。
並列摘要
Coxsackievirus A16 (CVA16) belongs to the Enterovirus genus of the Picornaviridae family, and is associated with herpangina and hand-foot-mouth disease. It may cause severe central neurological diseases and can be fatal. Yearly in Taiwan, children face the threats of CVA16 outbreaks during summer and autumn. However, to-date, no effective medication or vaccine against CVA16 infection exists, prompting an urgent need to develop effective antivirals for better management of CVA16 infection. In this study, we investigated the maximum tolerable concentrations of several natural compounds (as determined by XTT-based cell viability assay) in Rhabdomyosarcoma cell and tested their anti-CVA16 effect. Preliminary experiment indicated that two hydrolyzable tannins, Chebulagic acid (CHLA) and Punicalagin (PUG), exhibited highly efficacious antiviral activities. Specifically, the half-maximal cytotoxic concentration (CC50) of CHLA and PUG were 45.62 ± 9.58 and 76.96 ± 12.07 µM, respectively; the half-maximal effective concentration (EC50) of CHLA and PUG were 3.84 ± 0.91 and 4.61 ± 1.37 µM, respectively. Furthermore, CHLA and PUG exerted anti-CVA16 effects in a dose-dependent manner. To explore the underlying antiviral mechanism toward CVA16 life cycle, we employed a synchronized infection assay, which included pre-treatment, co-addition, and post-infection models, to identify their window of activity against viral entry, replication/translation, and release, respectively. Result showed that CHLA and PUG both disrupted viral infection during co-addition treatment, suggested that the tannins could potentially be mediating their anti-CVA16 effect via blocking viral entry. Further analysis is underway to clarify the tannins’ precise antiviral mechanism against CVA16 infection. In conclusion, we anticipate that these studies will help develop novel antiviral candidate for management against CVA16 infection.