在本實驗室之前研究發現虎杖根乙醇萃取物可以抑制 EB 病毒的溶裂循環，因此本研究探討虎杖根乙醇萃取物之分離物抑制 EB 病毒溶裂循環。將虎杖根乙醇萃取物以正己烷進行分劃，所得的分劃物，PcE(H) 再以乙酸乙酯溶劑進行分劃，得到的 PcE(H)EA 分劃物藉由半製備高效液相層儀分離出 F1、F2 及 F3 分離物；並且以逆相高效液相層儀分析各分劃物中白藜蘆醇及大黃素的含量。所得到的分劃物以 MTT 方法測定 P3HR1細胞存活率；以免疫墨點法、免疫螢光法及流式細胞儀分析 EB 病毒溶裂蛋白質的表現；以 Real-time PCR 方法分析病顆粒的釋放；以螢光素酶分析病毒溶裂循環基因的轉錄。結果顯示 PcE(H)EA 在濃度 6.3 g/ml 可以抑制 EB 病毒溶裂蛋白質的表現；而 F2 及F3 分別在濃度 12.5 及 6.3g/ml 時完全抑制 EB 病毒溶裂蛋白質的表現。而且F3 在濃度 6.3 g/ml時抑制病毒溶裂循環早期基因 BRLF1 轉錄活性，抑制率約 80%；也抑制了約 50% 病毒顆粒的釋放。此外，F3 成分中的大黃素為 68.2%，發現 F3和大黃素對 P3HR1 的細胞毒性濃度與抑制EB病毒溶裂白質的活性相類似，所以推測 F3 中的大黃素對 EB 病毒溶裂蛋白質的抑制作用扮演重要角色。 Before our laboratory studies found that ethanolic extract from Polygonum cuspidatum root (PcE) inhibits EBV lytic cycle. The study is to investigate that the inhibition of Epstein-Barr virus lytic cycle by fractions separated from Polygonum cuspidatum root. To partition the PcE with n-hexane, the polar layer named PcE(H), and then the PcE(H) partitioned by ethyl acetate, named PcE(H)EA. The PcE(H)EA was fractionated by semi-preparative high performance liquid chromatography (HPLC), named F1, F2 and F3. The contents of resveratrol and emodin in extracts and fractions were determined by HPLC. This investigation used MTT assay to determine the viability of P3HR1 cells. Immunoblot, indirect immunofluorescence, flow cytometry analyses were performed for the expression of EBV lytic proteins. Real-time PCR used to measure the EBV virion producing. Transient transfection and luciferase analyses used to assess the transcription of EBV immediate-early genes. Results showed that 3.1 g/ml of PcE(H)EA effectively inhibit the EBV lytic proteins expression by 50%. Moreover, F2 and F3 at concentration of 12.5 and 6.3 g/ml, completely inhibit lytic proteins expression. Adding 6.3 g/ml of the F3 into the P3HR1 cells culture medium inhibit the activity of the BRLF1 promoter by 80% and reduced the numbers of viral particles by 50%. Furthermore, F3 contains 68.2% emodin, and the effect of cell viability and inhibition lytic proteins expression is similar to emodin. This results suggest that emodin maybe a main
compound in F3 involved in inhibiting EBV lytic cycle.
