探討十六種草藥對登革病毒2型 (DENV-2) NS3蛋白酶抑製效應之研究

Abstract

每年約有3.9億例登革熱感染，此病毒在全球熱帶和亞熱帶氣候地帶有著顯著的發病率和死亡率；而先驅登革熱候選疫苗的保護效果也不盡理想，這促使新抗登革熱藥物的開發顯得更迫切。篩選藥用植物尋找其有效的抗登革熱的治療化合物可作為抗登革熱的替代策略， 因為藥用植物普遍低副作用而且容易從自然界中取材。病毒NS2B-NS3蛋白酶特定地切割前體多聚蛋白 (稱為自我剪切的現象）來釋放病毒複製的關鍵個體病毒蛋白，使其成為一個開發抑製劑的可行目標。在這項研究中，植物材料使用由低至高極性之溶劑即氯仿，乙酸乙酯，甲醇和水依次萃取。登革熱2型之NS2B-NS3蛋白酶通過大腸桿菌表現系統以最佳條件進行大量表現，並通過Ni-NTA親和層析純化。NS2B-NS3蛋白酶通過37℃的溫育後，通過SDS-PAGE分析鑑定，以確認其催化切割的活性；隨後再檢測評估植物萃取物對蛋白酶的自我剪切活性之抑製作用。實驗中一共對64種來自16個藥用植物的萃取物進行了篩選，已檢測它們對蛋白酶是否有抑製作用。Artemisia annua（青蒿）乙酸乙酯萃取物，Momordica charantia（苦瓜藤）甲醇萃取物和Scutellaria baicalensis（黃芩）的氯仿萃取物對蛋白酶自我剪切之活性具有抑製作用而這些植物萃取物之抑製作用在劑量反應試驗中也顯示出對劑量的依賴性。於此同時，實驗也發現另有10種萃取物對蛋白酶活性造成獨特的變化。這些擁有對蛋白酶自我剪切活性之抑製作用的植物都值得進一步研究，以確定和研究這些植物的生物活性化合物和植物化學，因為它們可能成為登革熱的替代治療。

Dengue virus (DENV) is responsible for 390 million infections annually along with significant morbidity and mortality throughout tropical and subtropical regions of the world. The protective efficacy for the most advanced dengue vaccine candidate were not satisfactory resulting in the search for new anti-dengue agents that are economical has assumed more urgency than in the past. Screening medicinal plants for anti-dengue activities is an alternative strategy in order to find the potent therapeutic compounds because of their expected low side effects and their high accessibility in nature. The viral non-structural 2B and 3 (NS2B-NS3) protease complex specifically cleaves the precursor polyprotein (a phenomenon known as autocleavage) to release individual viral proteins that are crucial for virus replication and hence, making it a promising target for development of inhibitors. In this study, plant materials were sequentially extracted using solvents of increasing polarity namely chloroform, ethyl acetate, methanol and water. The NS2B-NS3 protease complex from the DENV serotype 2 was overexpressed in E. coli using the optimal condition and purified by metal-chelate affinity chromatography. Autocleavage activity of the protease complex was firstly determined by incubation at 37°C followed by SDS-PAGE analysis to confirm the isolation of catalytically active protease. Plant extracts were then assessed for inhibitory effects against the protease autocleavage activity. A total of 64 extracts from 16 plants has been screened for inhibitory effects on the protease. Ethyl acetate extract of Artemisia annua, methanol extract of Momordica charantia and chloroform extract of Scutellaria baicalensis were tested positive for inhibitory effects on the protease autocleavage activity and the inhibitory effects were found to be in dose-dependent manner. Meanwhile, another 10 extracts were found to cause unique changes to the protease autocleavage activity. The plants that possess inhibitory effects on the protease autocleavage activity are worth to be further investigated to identify and study the plant bioactive compounds and its phytochemistry as they might be advantageous as an alternative for dengue treatment.