石榴皮抗日本腦炎病毒功效之研究

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Title:	石榴皮抗日本腦炎病毒功效之研究 Anti-viral activity of Pericarpium granati against Japanese encephalitis virus
Authors:	呂鎧任
Contributors:	醫學檢驗生物技術學系碩士班
Keywords:	日本腦炎病毒;石榴皮;安石榴苷 JEV;Japanese encephalitis virus;Pericarpium granati;Punicalagin
Date:	2011-07-29
Issue Date:	2011-10-17 16:56:52 (UTC+8)
Publisher:	中國醫藥大學
Abstract:	日本腦炎病毒(Japanese encephalitis virus, JEV)為正向單股RNA病毒，屬於黃病毒科病毒。日本腦炎病毒的感染大部分為無症狀感染，少部分輕微病例會產生頭痛、發燒、昏迷、痙攣、無菌性腦膜炎等症狀或死亡。日本腦炎的治療仍以支持療法為主，在目前尚未有有效的治療方法。石榴皮(Pericarpium granati, PG)煎劑是傳統中藥，常用於抗病毒，如在雞胚胎實驗中對流感病毒具有抑制作用，在細胞培養實驗也同樣具有抗疱疹病毒的能力。本研究探討石榴皮水萃物與石榴皮主成分Punicalagin抑制日本腦炎病毒感染細胞的功效及機轉。首先以MTT 試驗測得石榴皮水萃物對BHK-21細胞之半毒殺濃度(CC50)為53μg/mL，而對TE-671細胞之CC50為219.7μg/mL，在Punicalagin對BHK-21細胞的CC50為14μM，以及對TE-671細胞的CC50為42μM。石榴皮水萃物與Punicalagin具有劑量依賴模式抑制日本腦炎病毒感染BHK-21與TE671細胞株的細胞病變現象(Cytopathic Effect Assay ,CPE assay)。分別以先給藥一小時、先感染一小時或同時給藥與感染的條件下，以病毒蝕斑試驗(Plaque assay)計算抑制病毒斑點百分之五十的藥物濃度，發現石榴皮水萃物在先給藥再感染模式之藥物抑制日本腦炎病毒其病毒半抑制藥物濃度(IC50)為4.4μg/mL，同時給藥並感染模式之IC50為5.2μg/mL，而先感染後給藥之抑制日本腦炎病毒IC50則為74.6μg/mL；同樣地Punicalagin先給藥後感染的IC50為1.17μM，同時給藥感染模式的IC50為3.27 μM，先感染後給藥模式的IC50為9.18μM。由此結果指出，石榴皮水萃物與Punicalagin對日本腦炎病毒具有預防病毒感染細胞的能力。而在免疫路徑驗證的實驗中，發現NF-κB 蛋白會隨著石榴皮水萃物與指標成分Punicalagin濃度增加而增加其表現量，因此證明石榴皮水萃物與指標成分Punicalagin以會誘導細胞產生免疫發炎因子NF-κB的這條訊息路徑產生免疫反應抵抗病毒的感染。 Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus with positive-sense single-stranded RNA genome. JEV infection is asymptomatic, only few cases have headache, fever, encephalitis, coma and even death. Treatment for JE is supportive and still lack of efficacy. Water extrats of Pericarpium granati (PG) is an traditional herbal therapeutic with antiviral activities. It was shown to inhibit influenza A virus replication in chicken embryos and herpes simplex viruses in cell culture. In this study, we intend to investigate the anti-JEV activities and functional mechanism of PG water extract and the related compound punicalagin (PN). CC50 values (50% cytotoxicity concentration) of PG water extract by MTT test were 53 μg/mL for BHK-21 cells and 219.7 μg/mL for TE-671 cells, while CC50 values of PN were14 μM for BHK-21 cells and 42.7 μM for TE-671 cells. PG water extract and PN showed a concentration-dependently inhibitory effect on JEV-infected cytophatic effect and virus production in BHK-21 and TE-671 cells. The plaque reduction abilities of PG water extract and PN were studied by three different procedures including addition at 1 hour before virus inoculation (pre-treatment), addition and virus inoculation at the same time (simultaneous treatment), and addition at 1 hour after virus inoculation (post-treament). IC50 values (50% inhibition concentration) of PG water extract were 4.4 μg/mL by pre-treatment, 5.2 μg/mL by simutaneous treatment and 74.6 μg/mL by post-treament. IC50 values (50% inhibition concentration) of PN were 3.27 μM by pre-treatment, 1.17 μM by simutaneous treatment and 9.18 μM by post-treament. In vivo signaling pathway assay indicated PG water extract and PN inducing activation of the NF-κB signaling pathway.
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