Study of the Anti-Viral Effect and Its Mechanism of the Fucose and Sulfate Content from Brown Seaweed Polysaccharide Extracts on Dengue Virus Type II (Dv2) Infection

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Project title

Code/計畫編號

MOST105-2311-B034-001

Translated Name/計畫中文名

探討褐藻多醣萃取物之硫酸根與岩藻醣含量對第二型登革病毒之抗病毒效能及機制研究

Funding Organization/主管機關

National Science and Technology Center for Disaster Reduction

Co-Investigator(s)/共同執行人

詹伊琳(計畫主持人)

Department/Unit

Department of Life Science Chinese Culture University

Website

https://www.grb.gov.tw/search/planDetail?id=11875442

Year

2016

Start date/計畫起

01-08-2016

Expected Completion/計畫迄

31-07-2017

Co-Investigator(s)

Chang-Jer Wu

Bugetid/研究經費

750千元

ResearchField/研究領域

食品科技(工)

登革病毒為一個具有醣蛋白外套膜的單股正向 RNA 病毒，台灣在過去曾發生過三次大流行，顯示登革病毒感染在台灣為重要的流行性疾病。登革病毒感染會引起嚴重的臨床症狀，但其致病機制尚為完全被了解，亦尚無有效的登革病毒疫苗或藥物來預防人類感染。近年來有越來越多的研究顯示出藻類硫酸多醣具有抗氧化、抗發炎、抗病毒、抗癌、抗凝血等作用，為具抗登革病毒潛力之天然物。因此本計劃將以褐藻多醣萃取物評估其抗登革病毒效能，並進一步探討硫酸根和岩藻醣組成對於抗登革病毒效果是否具有關連性。於今年執行的計畫中，我們已從台灣附近海域的海藻中選出馬尾藻、昆布以及裙帶菜等三種褐藻，以熱水萃取的方式製備出褐藻多醣萃取物，並且進行其多醣組成的分析，並利用BHK-21 為細胞模式，於病毒吸附感染過程之不同時間點添加褐藻多醣萃取物，利用病毒斑抑制率試驗評估褐藻多醣萃取物抑制登革病毒吸附感染的效果，於初步的實驗中得到了以下結果：1. 褐藻多醣萃取物中，馬尾藻多醣硫酸根含量最多，而岩藻醣含量以昆布多醣最高；2. 褐藻多醣萃取物在病毒斑形成抑制率試驗中，以病毒感染細胞前加入抑制效果最好，其中抗登革病毒的活性以昆布多醣最佳。本計畫將延續前述之結果，利用細胞及動物實驗模式，對於其相關的機制進行更深入的探討，第一部分將利用in vitro 細胞相關實驗分析其抗病毒作用，以 BHK-21 cells 為細胞模式，於病毒吸附感染過程之不同時間點添加褐藻多醣萃取物，以免疫螢光染色觀察褐藻多醣萃取物是否可以降低登革病毒抗原於細胞內的表現量，最後藉由抽取細胞總 RNA 含量與 Real time PCR 的方式，分析於添加三種不同褐藻多醣萃取物後，其細胞內登革病毒之基因表現量，並探討相關的機制。第二部分為 in vivo 小鼠試驗，利用 C3H/HeN 品系的乳鼠建立登革病毒感染小鼠之動物模式，進行存活率觀察，並將小鼠犧牲進行腸胃道出血情形觀察、血液生化分析、Real time PCR 與組織病理切片，最後，將in vitro 及 in vivo 實驗的結果相互整合比較，探討褐藻多醣萃取物之抗登革病毒效果，及萃取物中硫酸根與岩藻醣組成對於其保護效果之影響。希望在未來二年的計畫能發展出有效的抗登革病毒感染的硫酸多醣萃取物，以作為抗登革病毒感染之保健食品應用。 Dengue virus (DENV) is a positive, single-stranded RNA virus with glycoprotein envelope, which caused three outbreaks in Taiwan. DENV infection has been shown an important epidemic disease in Taiwan. Mechanisms of how DENV causes clinical symptoms are not fully understood. There is still lacking of effective vaccines or drugs to prevent human from DENV infection. However, it has been shown that algae sulfated polysaccharides have effects of anti-oxidation, anti-inflammation, anti-virus, anti-cancer, anti-coagulation. With these effective properties, algae sulfated polysaccharides are considered as a potential anti-viral natural product nowadays. Therefore, we are going to investigate whether sulfated polysaccharide extracts from brown algae have anti-DENV effect and further evaluate the correlation between contents (sulfate and fucose) and anti-viral efficacy. In the ongoing project, we prepared polysaccharide extracts from three kinds of brown algae (Sargassum cristaefolium, Laminaria japonica, and Undaria pinnatifida) around Taiwan sea area by using hot-water extraction methods and analyze their components of polysaccharides. We also performed in vitro experiments to evaluate the inhibition effect of polysaccharide extracts in viral adsorption by using plaque reduction assay on BHK-21 cells. Polysaccharide extracts were added at different stages of viral adsorption to clarify their inhibitory effect of viral infection. The result showed that polysaccharides of Sargassum cristaefolium had the highest sulfated level, and those of Laminaria japonica contained the most fucose. Data also showed that brown algae extracts has the most viral inhibitory effect when added before viral adsorption in the plaque reduction assay. Comparing of three brown algae extracts, polysaccharides of Laminaria japonica had the highest anti-viral effect of DENV. In the extension of previous described results, project will investigate the anti-viral effect of DENV of brown algae extracts in both in vitro and in vivo systems. Firstly, the anti-viral effect of polysaccharides of brown algae will be performed in BHK-21 cells. Immunoflurorecent assay (IFA) will be used to investigate whether brown algae extracts can reduce the cellular expression levels of DENV antigens. Cellular RNAs will be prepared from samples treated with sulfated polysaccharide extracts during viral infection for further real-time PCR to evaluate the gene expression level of DENV. The mechanisms of anti-DENV effect will also investigate. Secondly, C3H/HeN suckling mice will be used to set up a DENV infection animal model for in vivo studies. With this part of experiments, survival rates, bleeding of gastrointestinal tract, biochemical analysis of blood, real-time PCR of tissues, and pathological examinations will be observed. Taken together, results of in vitro and in vivo studies will be compared and discussed to clarify the roles of polysaccharide extracts in anti-DENV abilities. In the future two years, we hope to develop the algae sulfated polysaccharides which may be used as a health food supplement of anti-DENV agents.

Keyword(s)

登革病毒
藻類硫酸多醣
褐藻
硫酸根
岩藻醣
感染小鼠模式
Dengue virus
Algae sulfated polysaccharides
Brown algae
Sulfate
Fucose
Infected mice model

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Study of the Anti-Viral Effect and Its Mechanism of the Fucose and Sulfate Content from Brown Seaweed Polysaccharide Extracts on Dengue Virus Type II (Dv2) Infection

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