虎杖是一種在亞洲廣泛使用的中草藥。先前在我們實驗室研究發現，虎杖根乙醇萃取物 (PcE) 可以抑制EB病毒的溶裂循環。進一步經由正己烷及乙酸乙酯分劃，所得分劃物，PcE(H)EA，藉由半製備高效液相層析儀 (HPLC) 分離純化得到F1、F2和F3分離物。F3主要成分是大黃素，含量為68.2%；F3和大黃素都能抑制EB病毒溶裂循環，抑制EB病毒溶裂蛋白質表現的EC50分別為5.9 g/ml及3.9 g/ml，所以證明参與F3抑制EB病毒溶裂循環的主要成分是大黃素。本研究主要探討虎杖根部乙酸乙酯分離物F1及F2對EB病毒溶裂循環的抑制影響。以MTT方法測定F1或F2對P3HR-1的細胞存活的影響。以西方墨點法、免疫螢光法及流式細胞儀分析EB病毒溶裂期蛋白質的表現。結果顯示F1抑制EB病毒溶裂期蛋白質的EC50是23.3 g/ml；F2則為7.8 g/ml ，而對P3HR1細胞毒性，F1及F2的CC50分別是93.1 g/ml及6.1 g/ml。所以推測F2對溶裂蛋白質的抑制作用可能是細胞毒性所造成的影響，而F1在不具細胞毒性濃度能有效抑制EB病毒溶裂期蛋白質表現。以Real-time quantitative PCR方法分析F1對EB病毒DNA的複製與病毒顆粒的釋放影響，結果顯示F1抑制EB病毒DNA的複製，EC50為55.5 mg/ml，而且在濃度50 g/ml可以減少病毒顆粒的釋放達44.5 %。本研究結果證實，F1能有效的抑制 EB 病毒的溶裂蛋白質表現，使EB病毒DNA複製能力受到抑制，而降低了病毒顆粒的釋放量。 Polygonum cuspidatum is widely used as a medicinal herb in Asia. From the results of our previous studies, we found that ethanolic extract from the Polygonum cuspidatum root (PcE) inhibits EBV lytic cycle. The PcE was partitioned with n-hexane and ethyl acetate, and then PcE(H)EA was fractionated and purified by semi-preparative high performance liquid chromatography (HPLC) into F1, F2 and F3. F3 contained 68.2% emodin. F3 and emodin inhibit EBV lytic cycle, the concentration of F3 and emodin required to inhibit EBV immediate-early protein expression by 50% are 5.9 g/ml and 3.9 g/ml, respectively. These results demonstrated that emodin is a main compound involved in inhibition of F3 on EBV lytic cycle. The purpose of this study is to investigate the inhibitory effects of the ethyl acetate subfractions, F1 and F2 from Polygonum cuspidatum root on EBV lytic cycle. MTT assay was used to determine the viability of P3HR1 cells. Immunoblot, indirect immunofluorescence, flow cytometry analyses were performed for the determining of the expression of EBV lytic proteins. Results showed that the concentration of F1 and F2 required to inhibit EBV immediate-early protein expression by 50% are 23.3 g/ml and 7.8 g/ml, respectively. F1 and F2 decrease cell viability to 50% (CC50) at 93.1g/ml and 6.1 g/ml, suggesting that F2 inhibited the expression of EBV lytic proteins was due to cell toxicity, F1 inhibits the expression of EBV lytic protein at the concentration of no cytotoxicity toward P3HR1 cells. Real-time quantitative PCR was used to analysis the replication of EBV DNA and the EBV virion producing, showing that F1 inhibited the replication of EBV DNA, EC50 is 55.5 g/ml, reduces virion production by 44.5 % at the concentration of 50 g/ml. This study finish that F1 inhibits EBV lytic proteins expression, thereby impeds the replication of EBV DNA and production of mature viral particle.
