| Summary: | 碩士 === 高雄醫學大學 === 醫學研究所碩士班 === 93 === A worldwide outbreak of severe acute respiratory syndrome (SARS) which might originate from Guangdong, China, had spread into many countries including Taiwan in 2003. A novel coronavirus (SARS-coronavirus, SARS-CoV) is the causative agent. To seek potential candidate drugs for treating SARS, we screened a total of 1022 samples including 735 crude extracts and 287 pure compounds from traditional Chinese medicinal herbs and Taiwanese plants. Instead of SARS-CoV, the human coronavirus 229E (HCoV-229E) was used to study the antiviral activities of the samples by using the XTT assay kit. The SARA-CoV protease inhibition activity was studied using recombinant SARS-CoV protease 3CLpro with 3 kinds of proteomic system (in vitro). The inhibition of the samples on the ICAM-1 expression was assayed using A549 cells which had been activated by HCoV-229E-infected supernatant. The results of antiviral activity test revealed that a total of 19% (197/1022) including 156 crude extracts (50µg/ml) and 41 pure compounds (5µg/ml) possessed antiviral activity against HCoV-229E of more than 50%. Eight of the 50 potential candidates showed >10% inhibition of azocasein cleavage by SARS-CoV protease. Nine of the 15 potential anti-SARS-CoV protease candidates showed >50% inhibition rate in trans-cleavage assay. Three of the 35 potential candidates showed >50% inhibition of cis-cleavage by SARS-CoV protease in Vero cells. The ICAM-1 expression on lung cancer cells A549 were activated by HCoV-229E-infected supernatant. The activation was blocked by thirty one of the 582 samples tested (5%) including 14 crude extracts and 17 pure compounds. Seven of the 14 potential antiviral candidates (50%) exhibited the direct inhibition of SARS-CoV replication. In traditional Chinese medicines, Ge-Gen-Tang (GGT) and Xiao-Qing-Long-Tang (XQLT) are two common remedies used for treating cold and flu. However, neither of these formulas has been reported with the scientific evidence to prove their specific efficacy in treating and preventing HCoV-229E infections. Therefore, in this study, we firstly measured the antiviral activities of these two formulas on HCoV-229E infected MRC-5 cells, normal human fetal lung fibroblasts, by using XTT assay kit. The antiviral activity of IC50 was defined as the concentration achieved 50% cytoprotection against virus infection. The results showed that the IC50 values of the crude extracts from GGT and XQLT against HCoV-229E infection were 31.71µg/ml and 110.73µg/ml, respectively. The concentration of GGT and XQLT that induced 50% cytotoxicity (CC50) to MRC-5 cells were 1718.9µg/ml and 457.46µg/ml, respectively. The selectivity index ( SI: CC50 / IC50 ) , for GGT was 54.21 and for XQLT was 4.13, which indicated that these two Chinese medicines have direct antiviral activities against HCoV-229E . Further time course studies were carried out to elucidate the mechanism of how the drugs inhibit HCoV-229E infection. The results showed that GGT and XQLT exhibited preventive and therapeutic effects. The antiviral attachment activity of IC50 values for GGT and XQLT were 271.2 µg/ml and 70.8 µg/ml, respectively. The antiviral penetration activity of IC50 value, for GGT was 98.12µg/ml and for XQLT was 12.4µg/ml. The above evidences have proven that these Chinese medicines are effective in treating and preventing cold and flu caused by HCoV-229E infections. But, could GGT and XQLT defeat cold and flu caused by other respiratory viral infections? And, what are the mechanisms? Further study is necessary to answer these questions.
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