先前研究發現虎杖根乙醇萃取物會導致EB 病毒腫瘤細胞死亡及在濃度25 μg/ml 抑制丁酸鈉所誘發EB 病毒早期基因的轉錄及溶裂蛋白質的表現，進而抑制病毒顆粒產生。進一步分析虎杖根乙醇萃取物的成分，發現大黃素及白黎蘆醇分別佔萃取物成份 2.48%及0.6%；在9.37 (2.53 μg/ml)及55 μM (12.55 μg/ml)才有明顯抑制病毒顆粒產生的能力。所以推論虎杖根乙醇萃取物不完全藉由大黃素及白黎蘆醇達到抗EB 病毒的功效。此外, 由先驅試驗發現虎杖根乙醇萃取物具有抑制EB 病毒表現latent membrane protein 1 (LMP1)； LMP1 是致癌蛋白質，能引發EB 病毒細胞增殖及轉型成腫瘤；因此推測虎杖根乙醇萃取物應該含有抗EB 病毒及抗腫瘤的有效成分。所以本計劃分三部份：第一部份、抗病毒及抗腫瘤有效成份的分離及鑑定。第二及第三部份、探討抗EB 病毒及毒殺EB 病毒淋巴腫瘤細胞有效成分之作用機制。我們將以矽膠薄層及逆相式高效液相色層分析法將抗EB 病毒及抗腫瘤有效成分分離，並且進行鑑定。以西方墨點法及real-time PCR 分析抗EB 病毒有效成份對病毒溶裂蛋白質表現及定量EB 病毒DNA，以了解有效成份抑制EB 病毒感染、複製及病毒顆粒形成。以細胞毒性實驗及細胞核型態學觀察探討有效成分抑制腫瘤細胞生長的影響，也以彗星膠體電泳法分析有效成份引發腫瘤細胞核內DNA 片段化的影響，進而以流式細胞儀定量凋亡的細胞數。並且以免疫螢光法分析抗腫瘤的有效成分是否抑制腫瘤細胞內NF-κB 的表現。這個研究將有助於更了解虎杖根抗病毒及抗腫瘤有效成分的作用機制。 From our recent studies demonstrate that the ethanolic extract of Polygonum cuspidatum has cytotoxicity toward Epstein-Barr virus (EBV)-positive tumor cells and effectively inhibit the transcription of BRLF1 and BZLF1, two EBV immediate-early genes that encode Rta and Zta, respectively and inhibit the expression of lytic proteins, Rta, Zta and EA-D, during the viral lytic cycle in P3HR1 cells at the concentration of 25 μg/ml. This study finds that the inhibition prevents the virus from producing mature viral particles. Further analysis for the constituents of the ethanolic extract of Polygonum cuspidatum; found that emodin and resveratrol occupy the extract ingredient 2.48% and 0.6% respectively. Emodin of 9.37 (2.53 μg/ml) and resveratrol of 55 μM (12.55 μg/ml) inhibit the virion producing. Therefore, I suggest that bioactive components of anti-EBV activity from the ethanolic extract of Polygonum cuspidatum may be not only mediated by emodin and resveratrol. In addition, based on the preliminary studies, the ethanolic extract of Polygonum cuspidatum also displays the inhibitory effect on the expression of latent membrane protein 1 of EBV. LMP 1 is an oncoprotein, confers the ability of proliferation and transformation for EBV-infected cells to tumor cells, Therefore, I propose that the ethanolic extract of Polygonum cuspidatum contains the bioactive components of anti-EBV and anti-tumor activity. In this proposed study, I will to perform three parts: Part I, to isolate and identify the antiviral and anti-tumor components. Part II and Part III to investigate the mechanism of anti-EBV and cytotoxicity to EBV-positive tumor cells. Thin layer chromatography and reverse phase high performance liquid chromatography will be used to isolate and identify the antiviral and anti-tumor components. Using western blotting and real-time PCR to study the expression of EBV lytic proteins and determine the amount of EBV DNA, these results will be promoted to understand bioactive components to EBV infection, replication and virus particle production. I will perform the cytotoxicity assay and nuclei morphology observation was used to analyze whether bioactive components have the anti-proliferative activity on EBV-positive tumor cells. Comet assay was used to analyze the bioactive components induced DNA fragmentation of EBV-positive tumor cells. Indirect immunofluorescence was performing to investigate the NF-κB expression. The study is important for elucidating the antiviral and antitumor mechanism of Polygonum cuspidatum.
