| 摘要: | 本研究主要目的是從蛇總管(Sida acuta)、穿心蓮(Andrographis paniculata)、白鶴靈芝(Rhinacanthus nasutus)、虎杖(Polygonum cuspidatum)、仙草(Mesona chinensis)、角菜(Artemisia lactiflora)、毛梗豨簽(Siegesbeckia glabrescens)水和乙醇萃取物中篩選具有抗EB病毒效應的中草藥。本實驗是以光化學方法測定萃取物的總酚類含量；利用西方墨點法分析萃取物是否抑制 EB 病毒溶裂蛋白質－Rta、Zta及EA-D 的表現；而細胞毒性試驗則是以MTT的方法分析；對早期基因轉錄的影響是以螢光素酶活性進行分析；以PCR及Real-time PCR方法偵測EB病毒的DNA含量，以了解是否抑制EB病毒複製及病毒顆粒產生。結果發現在總酚類含量測定以虎杖乙醇萃取物含量為最高。抗病毒活性的篩選上發現穿心蓮、虎杖、仙草在100 g/ml 對EB病毒溶裂蛋白質－Rta表現分別有58.4、100.0、66.3%的抑制率；進一步也發現穿心蓮的乙醇萃取物濃度在25g/ml 時抑制Rta、Zta 及EA-D表現的能力高於50%，而且對細胞不具明顯的毒性。在穿心蓮主要的成分-穿心內酯 (Andrographolide)濃度5 g/ml 時對細胞不具有毒性，對EB病毒溶裂蛋白質－EA-D的表現有84.3 ± 15.7%的抑制率。穿心蓮乙醇萃取物25 g/ml或穿心內酯5 g/ml對EB 病毒BRLF1與BZLF1基因轉錄具有抑制作用，前者抑制率分別為78.7 ± 2.7%及82.6 ± 5.1%，後者分別為61.9 ± 5.0%與92.4 ± 0.6%。Real-time PCR的結果顯示，添加穿心蓮乙醇萃取物與穿心內酯與只加入誘導劑相比，降低1.1-1.5 log值。由這些結果證明穿心蓮乙醇萃取物與穿心內酯可以有效抑制EB病毒早期基因的轉錄，且影響EB病毒溶裂蛋白質表現，進而抑制病毒複製與病毒顆粒的釋放而達到抗病毒的效果。
This investigation examines the aqueous and ethanolic extract from Andrographis paniculata, Rhinacanthus nasutus, Polygonum cuspidatum, Mesona chinensis, Artemisia lactiflora, and Siegesbeckia glabrescens for their anti-Epstein Barr virus (EBV) activities. This work uses a photochemical method to determine the total content of phenolic compounds in the extracts; immunoblotting, inhibition of the expression of EBV immediate-early and early proteins, Rta, Zta and EA-D; the MTT [ 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay] method, cytotoxicity; transient transfection assay, inhibition of the transcription of EBV immediate-early genes, BRLF1 and BZLF1; real-time PCR, EBV virion production. The results showed that the ethanolic extract from P. cuspidatum contains the highest amount of polyphenols. At 100 g/ml, the ethanolic extract from A. paniculata, P. cuspidatum, and M. chinensis inhibit the expression of Rta by 58.4%, 100.0%, and 66.3%, respectively. Furthermore, at 25 g/ml, the ethanolic extract from A. paniculata inhibits the expression of Rta, Zta, and EA-D by more than 50% but exhibits little cytotoxicity. Furthermore, the major component in A. paniculata, andrograpolide, at 5 g/ml, not only inhibits the expression of EA-D by 84.3 ± 15.7% but also exhibits little cytotoxicity. Meanwhile, either 25 g/ml of ethanolic extract from A. paniculata or 5 g/ml of andrographolide inhibits the transcription of BRLF1 by 78.7 ± 2.7% and 82.6 ± 5.1% ; BZLF1, 61.9 ± 5.0% and 92.4 ± 0.6% , respectively. Real-time PCR revealed that the ethanolic extract from A. panuculata and andrograpolide inhibit the production of EBV particles by about 10- to 15-fold. These results demonstrate that ethanolic extract from A. panuculata and andrograpolide effectively inhibit the transcription of EBV immediate-early genes and ultimately influence the expression of EBV lytic proteins to inhibit the EBV lytic cycle. |
