螺旋藻抽取液Apogen對抗病毒感染之作用探討

Abstract

輪狀病毒和41型腺病毒為造成小兒急性腸胃炎之病毒，呼吸道融合病毒及第三型副流感病毒為造成小兒下呼吸道疾病之病毒，A24型克沙奇病毒變異病毒株造成的疾病為急性出血性結膜炎。至目前為止，針對以上五種病毒引起的疾病目前仍無有效的治療方式。由於先前已有研究指出本次實驗使用的螺旋藻(Spirulina platensis)抽取液商品Apogen對於71型腸病毒感染造成的細胞凋亡具有抑制作用。也有其他研究指出螺旋藻具有抗病毒的能力。因此本研究希望能瞭解螺旋藻抽取液是否對於這些病毒的感染同樣具有抑制效果。在本篇論文中，分別利用螢光焦點減少試驗、MTT細胞活性試驗及血球吸附試驗得到Apogen能抑制50%以上輪狀病毒、呼吸道融合病毒及第三型副流感病毒感染之藥物濃度(EC50)分別為0.327 mg/ml、0.195 mg/ml及1.17 mg/ml。並利用MTT試驗得到50%細胞毒性濃度(CC50)，最後經計算可得到Apogen對輪狀病毒、呼吸道融合病毒及第三型副流感病毒之選擇性指標(SI)分別為55、98及12.9。從研究結果中也發現Apogen對A24型克沙奇病毒變異病毒株及41型腺病毒並無抑制效果。本研究亦測試了Apogen中的藻膽色素蛋白質成份，包括異藻膽蛋白質(APC)及藻膽蛋白質(CPC)，結果顯示所測試的病毒並不能被藻膽色素蛋白質抑制。透過藥物作用時間點試驗，發現Apogen能抑制呼吸道融合病毒及第三型副流感病毒進入細胞，也能抑制輪狀病毒於細胞中的生長。透過細胞結合及細胞穿透抑制試驗瞭解Apogen能抑制呼吸道融合病毒及第三型副流感病毒與細胞之結合以及穿透細胞。在第三型副流感病毒部分，利用血球凝集抑制試驗及讓細胞表現病毒HN蛋白質的方式進行血球吸附抑制試驗，結果發現Apogen並不會影響血球凝集素的活性。輪狀病毒部份，從凝膠電泳以及即時聚合酶連鎖反應的結果發現病毒RNA的複製並不受Apogen影響，但從螢光抗體染色的結果發現病毒蛋白質的合成有被Apogen抑制的現象。以上結果顯示Apogen中可能存在蛋白質以外的物質能抑制輪狀病毒、呼吸道融合病毒及第三型副流感病毒之生長，且抑制的成份可能不只一種或同時具有多重抑制功能。之後希望能分離出Apogen中真正能有效抑制病毒的成份，並進一步探討藥物抗病毒之機制。

Rotavirus and adenovirus 41 (Ad41) are the etiological agents of acute gastroenteritis in infants and children, respiratory syncytial virus (RSV) and parainfluenza virus 3 (PIV3) are the etiological agents of lower respiratory tract infectious diseases in infants and children, and coxsackievirus A24 variant (CA24v) causes acute conjunctivitis. There are still no effective treatment for the infection of these viruses. Previously, studies have shown that enterovirus 71 induced cell apoptosis could be inhibited by commercial extracts of blue-green alga, Spirulina platensis “Apogen”, and the antiviral activity of Apogen against other viruses has also been demonstrated. In this study, we intend to understand whether the “Apogen” has antiviral activity against these viruses. Fluorescent focus reduction assay, MTT test, and hemadsorption test were used to determine the EC50 (effective concentration 50%) for rotavirus, RSV and PIV3, respectively, and the values were 0.327 mg/ml, 0.195 mg/ml and 1.17 mg/ml, respectively. The MTT test was also used to evaluate the CC50 (cytotoxicity concentration 50%), and the SI (selectivity index) was calculated. The SI values of rotavirus, RSV, and PIV3 are 55, 98, and 12.9, respectively. We also found that Apogen could not inhibit CA24v and Ad41 infection. The antiviral activities of the major protein components of Apogen, allophycocyanin and C-phycocyanin, were also evaluated and no antiviral activities were found. By the time of drug addition test, we found that Apogen could inhibit the infection of RSV and PIV3 and the replication of rotavirus in cells. The inhibition test of cell attachment and penetration provided the evidence that Apogen could inhibit RSV and PIV3 binding to cells and penetrating into cells. For PIV3, by using the hemagglutination inhibition test and hemadsorption inhibition test using HN expressed cells, we found that the activity of hemagglutinin was not influenced by Apogen. For rotavirus, by using RNA PAGE, real-time PCR, and fluorescent antibody staining, we found that the viral protein synthesis was inhibited by Apogen, but the viral RNA replication not. In conclusion, the results indicated that Apogen contains one constituent with multiple antiviral functions or two or more constituents exhibiting different antiviral activities and the substances may not be proteins. In the future, we hope to isolate the real antiviral components and further investigate the antiviral mechanisms.