傳統中藥方劑及台灣本土植物之抗冠狀病毒(人類冠狀病毒229E及SARS冠狀病毒)和抗發炎之研究

Study of traditional Chinese medicines and Taiwanese herbs for biological activities against coronavirus (human coronavirus 229E and SARS coronavirus ) and inflammation

楊易靜(I-Ching Yang)

指導教授 ： 林貴香 ； 蔣連財

高雄醫學大學/醫學院/醫學研究所/碩士(2005年)

摘要

2003年由中國廣東爆發了嚴重急性呼吸道症候群(Severe Acute Respiratory Syndrome;SARS)疫情並引起世界性的恐慌， 一種全新的人類冠狀病毒SARS-CoV為此次疫情的元兇。為了尋求治療SARS病毒之藥物，我們篩檢了1022種來自中草藥之粗萃取物(735種)及純化合物(287種)。篩檢方法為(1)初期以229E人類冠狀病毒(Human coronavirus 229E;HCoV-229E)為篩檢對象，來代替SARS-CoV，測定XTT還原產物量，以偵測藥物之抗病毒活性。(2)測定抑制SARS-CoV 增殖活性。(3)以SARS-CoV之Protease重組蛋白3CLpro為標的，並分別作三種in vitro測試，以偵測藥物對Protease之抑制活性。 (4)測試藥物阻斷經HCoV-229E感染MRC-5細胞之上清液處理之A549細胞之重要發炎因子ICAM-1表現之能力。研究結果發現：(1) 有19% (197/1022)種樣品, 包括156種粗萃取物(50µg/ml)及41種純化合物(5µg/ml)，在此低濃度時有大於50％之抗HCoV-229E病毒感染活性。(2)直接測定抑制SARS-CoV 增殖活性的陽性結果為50%（7/14）。(3)在50種粗萃取物中有8種具10%以上的SARS-CoV重組Protease抑制活性（Azocasein-cleavage）。15種粗萃取物中有9種具50%以上的SARS-CoV重組Protease抑制活性( Trans-cleavage )。35種粗萃取物中有3種具50%以上的重組Protease抑制活性（Cis-cleavage）。(4)肺癌細胞A549之ICAM-1表現可被HCoV-229E感染細胞的上清液所活化，此活化現象可被5%（31/582）的測試樣本所抑制。葛根湯及小青龍湯在中國傳統醫學為兩種常用之治療感冒中藥方劑，然而在已知的相關文獻報告中，卻缺乏直接的科學證據來顯示是否此兩種方劑有直接的抑制229E人類冠狀病毒感染作用;因此本篇研究首先以此兩種方劑，利用正常人類胚胎肺纖維母細胞MRC-5細胞感染HCoV-229E病毒，根據XTT方法來偵測藥物抗病毒活性，結果發現葛根湯及小青龍湯其抑制病毒感染50 %的劑量(IC50)分別是31.71µg/ml 和110.73µg/ml，而對細胞毒殺50 % 的劑量(CC50)分別是1718.9µg/ml和457.46µg/ml。 兩種藥方的選擇指數(Selectivity index : CC50 / IC50)在葛根湯為54.21，在小青龍湯為4.13，證實了此兩種方劑可直接抑制229E人類冠狀病毒感染的治療效果;為了了解藥物對抑制229E人類冠狀病毒感染之作用機轉，接著做了時間效應(Time course)實驗，發現兩種方劑均有預防和治療雙重功效;進一步作抗病毒機轉研究，結果顯示：葛根湯抑制病毒附著之IC50值為271.2µg/ml，而阻止病毒入侵感染之IC50值為98.1µg/ml；小青龍湯抑制病毒附著之IC50值為70.8µg/ml，而阻止病毒入侵感染之IC50值為12.4µg/ml，但是葛根湯及小青龍湯對其它的感冒病毒是否有效？ 其作用機轉又是如何？ 尚待將來更進一步的研究結果來了解。

關鍵字

肺纖維母細胞株 ； 冠狀病毒 ； 中草藥

並列摘要

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.

並列關鍵字

ICAM-1 ； MRC-5 ； 3CLpro ； Human coronavirus ； Chinese herb

參考文獻

Akase T , Hamada Y , Higashiyama D , Akase T , Tashiro S , Sagawa, K., Shimada S : Trades in the prescriptions of kampo medicines over a six-years period. J Trad Med 19: 58-75, 2002.

Bacha U , Barrila J , Velazquez CA , Leavitt SA , Freire E : Identification of novel inhibitors of the SARS coronavirus main protease 3CLpro. Biochemistry 43(17): 4906-12, 2004.

Bermejo M , Jimenez JF , Munoz JL , Fernandez A : Pentoxifylline and severe acute respiratory syndrome (SARS): a drug to be considered. Med Sci Monit 9(6): SR29-34, 2003.

Bian YJ , Qi WS , Song QQ : Evaluation on effect of integrative medical treatment on quality of life of rehabilitation stage in 85 patients with SARS. Zhongguo Zhong Xi Yi Jie He Za Zhi 23(9): 658-60, 2003.

Blanchard JE , Elowe NH , Huitema C , Fortin PD , Cechetto JD , Eltis LD , Brown ED : High-throughput screening identifies inhibitors of the SARS coronavirus main proteinase. Chem Biol 11(10): 1445-53, 2004.