1998年,台灣地區爆發大規模腸病毒71型(EV71)的感染,當年造成十二萬名孩童受到感染,更有七十八例嚴重致死的病例,顯示研發抗病毒藥物的重要性,本研究是為了開發治療腸病毒71型的生藥資源。 本計劃挑選了葛根湯方劑及其單方做初步篩選進行實驗。葛根湯為中國傳統醫學常用之治療感冒方劑,在目前有研究報告指出葛根湯具有抗流感病毒與疱疹病毒(HSV-1)的活性,但仍缺乏科學證據來證明葛根湯有直接抑制EV71感染的作用,因此我們篩選了葛根湯及其單方共八種藥物,利用XTT分析法,確認其抗病毒活性,發現葛根湯及麻黃其抑制病毒感染50%的劑量(IC50)分別是1.02μg/ml和1.49μg/ml,兩種藥物造成50%的細胞毒性(CC50)的劑量皆大於3000μg/ml,選擇指數(Selectivity index;SI)分別是>2429和>2011,低IC50證明有效,高SI證明安全,証實這兩種藥物可以有效和安全的抑制EV71。 為了瞭解藥物抑制EV71之作用機轉,因此進行時間效應(Time course)實驗,發現對於感染前、感染後皆有抗病毒活性,因此推論兩種藥物均有預防及治療功效;再進一步利用麻黃的純化合物p-coumaric acid、quercetin和vanillic acid做抗病毒試驗:結果顯示p-coumaric acid的CC50> 100μg/ml,IC50為0.17μg/ml,SI>602.4,quercetin的CC50為50.58μg/ml,IC50為0.078μg/ml,SI為684.5,vanillic acid的CC50>100μg/ml,IC50為0.16μg/ml,SI>625,顯示三種純化合物有很好的抑制EV71的效果;為了瞭解麻黃純化合物p-coumaric acid抗EV71病毒的作用機轉,進行時間效應試驗:結果顯示,感染前、感染後皆有抗病毒活性,因此推論p-coumaric acid在預防及治療都有不錯的效果。為瞭解其感染前給藥有效的作用機轉而進行病毒附著試驗及病毒穿透試驗,結果顯示p-coumaric acid抑制病毒附著之IC50值為0.52μg/ml,而阻止病毒入侵感染之IC50值為0.045μg/ml。 上述實驗的結果顯示出葛根湯複方及其單方,具有被應用來治療EV71病毒感染的潛力,未來可深入研究其抑制感染後的抗病毒作用機轉,而本研究結果也可提供未來研發抗腸病毒71型藥物之參考。
In 1998, there was a large outbreak of enterovirus 71 (EV71) infection in Taiwan with more than 12000 victums and 78 murtalities. Most of the infected and the dead are child younger than 5 years old. Effective anti-EV71 agents are needed urgently. We tried to seek potential herbal candidate to traet EV71. GGT is a traditional Chinese herbal remedy and commonly used to treat infections diseases including colds and influenza. We would like to screen Ge-Gen Tang (GGT) and its related ingredients in inhibit EV71 infection. However, neither of GGT, nor its ingredients has been reported to have efficacy in treating or preventing EV71 infections. Therefore, we firstly measured the antiviral activities of these eight formulas used the XTT reduction assay. The IC50 of antiviral activity concentration achieved 50% cytopritect againt viral infection. The results showed that the IC50 of the crude extracts of GGT and Ephedra sinica Stapf. against EV71 infection were 1.02μg/ml and 1.49μg/ml, respectively. Both of the induced 50% cytotoxicicty (CC50) of GGT and Ephedra sinica Stapf. were larger than 3000μg/ml. The selectivity index (SI: CC50 / IC50) of GGT and Ephedra sinica Stapf. were more than 2429 and 2011, respectively. These results indicated that GGT and Ephedra sinica Stapf. have effective antiviral activities against EV71. Further. Our results showed that GGT and Ephedra sinica Stapf. exhibited their anti-EV71 both before and after EV71 infection. There, they had both preventive and therapeutic potential. After we proved that Ephedra sinica Stapf. had the anti-EV71 activity, we would like to know which pure compound in Ephedra had this activity.Then we used to repeat these experiments p-Coumaric acid、Quercetin dihydrant and Vanillic acid. The results showed that the IC50 values of the pure compounds from p-Coumaric acid、Quercetin dihydrant、Vanillic acid against EV71 infection were 0.17μg/ml, 0.078μg/ml, and 0.16μg/ml, respectively. The CC50 of p-Coumaric acid、Quercetin dihydrant、Vanillic acid were larger than100μg/ml ,58.58μg/ml, and greater than 100μg/ml, respectively. The selectivity index (SI: CC50 / IC50), of p-Coumaric acid was larger than 602.4 and for Quercetin dihydrant was 684.5, Vanillic acid was larger than 625. Time course studies were carried out to cletermine whether these agents worked before or after EV71 infection and showed that p-Coumaric acid exhibited preventive and therapeutic effects. The antiEV71 attachment activity IC50 of p-Coumaric acid was 0.52μg/ml. The antiviral penetration activity IC50 of p-Coumaric acid was 0.045μg/ml. Our results above have proven that GGT, Ephedra sinica Stapf. and several pure compounds are effective in treating and preventing EV71 infections. Further study is necessary to understand the mechanism of these agents.
為了持續優化網站功能與使用者體驗,本網站將Cookies分析技術用於網站營運、分析和個人化服務之目的。
若您繼續瀏覽本網站,即表示您同意本網站使用Cookies。