海洋真菌之幽隱炭團菌及草酸青黴菌的次級代謝物對EB病毒再活化的抑制

Abstract

Epstein-Barr病毒 (Epstein-Barr virus, EBV)為人類第四型疱疹病毒，會感染90 %以上的人，主要會感染B細胞與上皮細胞，具有潛伏期及溶裂期兩種生活史。EB病毒的再活化與許多癌症及自體免疫疾病相關，目前相關疾病的治療主要依賴Acyclovir這類型的廣效抗病毒藥物，其針對溶裂期DNA複製達到抑制效果，但由於此類藥物無法根除病毒潛伏，後續仍會因病毒再活化而造成疾病復發或惡化。因此，開發能夠抑制病毒再活化的新型藥物顯得尤為迫切。近年來，海洋生物在天然藥物的研究領域中逐漸受到重視，因海洋環境的獨特性，造就這些生物產生許多具有藥用價值的次級代謝產物。本研究透過海洋軟體動物文蛤 (Meretrix lusoria) 之衍生真菌作為標的，旨在尋找具有抑制EB病毒再活化的化合物。首先，從文蛤 (Meretrix lusoria) 分離出26株真菌，其中有5株所產生的萃取物具有顯著的抑制EB病毒Rta的表現，然後選定其中2株進行後續分析，經過物種鑑定，確認此二真菌分別為Annulohypoxylon stygium及Penicillium oxalicum。透過大量培養獲取更多次級代謝物，並以相同的萃取方法進行處理，最終分別得到713.4 mg及813.5 mg之粗萃物。接著，結合Sephadex LH-20管柱層析及逆相高效液相層析 (RP-HPLC) 進行分離純化，同時在分離過程中持續追蹤抑制病毒再活化效果，最終得到三個化合物，分別為2-呋喃甲醇 (2-furanmethanol) 、5-羥甲基-2-呋喃甲酸 (5-hydroxymethyl-2-furoic acid) 及2-呋喃甲酸 (2-furoic acid) 。其中，2-呋喃甲醇的藥理活性較顯著，EC50和CC50分別為7.49 µM 和125 µM，SI值達16.7，並且會抑制病毒的極早期蛋白質Zta及早期蛋白質EA-D的表現，在15 µM濃度下能夠有效抑制病毒顆粒的生成。未來將聚焦於候選化合物之藥效機制，以期為新型抗EB病毒治療策略提供新的方向。

Epstein-Barr virus (EBV), also known as human herpesvirus 4, infects over 90% of the human population, primarily targeting B cells and epithelial cells with two distinct life cycle, latency and lytic phase. The reactivation of EBV is associated with numerous cancers and autoimmune diseases. Current treatments for related diseases mainly rely on broad-spectrum antiviral drugs like acyclovir, which inhibits lytic DNA replication. However, since these drugs cannot eradicate the latent virus, subsequent viral reactivation can lead to disease recurrence or exacerbation. Therefore, the development of novel drugs that can inhibit viral reactivation is particularly urgent. In recent years, marine Mollusca have gained increasing attention in the field of natural drug research. The unique marine environment has led these organisms to produce many secondary metabolites with medicinal value. This study targets the derived fungi of the marine Mollusca Meretrix lusoria, aiming to find compounds that inhibit EBV reactivation. Initially, 26 fungal strains were isolated from Meretrix lusoria. Among them, the extracts produced by 5 strains inhibited the expression of EBV Rta. Two of these strains were then selected for subsequent analysis. Through species identification, these two fungi were confirmed to be Annulohypoxylon stygium and Penicillium oxalicum. Mass cultivation was performed to obtain more secondary metabolites, which were processed using the same extraction method, yielding 713.4 mg and 813.5 mg of crude extracts, respectively. Then, separation and purification were conducted using Sephadex LH-20 column chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC), while continuously tracking the inhibitory effect on viral reactivation throughout the separation process. Three compounds were identified including 2-furanmethanol, 5-hydroxymethyl-2-furoic acid, and 2-furoic acid. Among them, 2-furanmethanol exhibited significant pharmacological activity, with an EC50 of 7.49 µM and a CC50 of 125 µM, resulting in a selectivity index (SI) of 16.7. 2-Furanmethanol also inhibited the expression of the viral immediate-early protein Zta and the early protein EA-D, and it effectively reduced the production of viral particles at a concentration of 15 µM. Future research will focus on the pharmacodynamic mechanism of the candidate compound, with the hope of providing a new direction for novel anti-EBV therapeutic strategies.