升麻葛根湯及其相關成份抗呼吸道融合病毒活性及其作用機轉之研究

Anti-respiratory syncytial virus activity and mechanism of Sheng-Ma-Ge-Gen-Tang and its related ingredients

王國治(Kuo-Chih Wang)

指導教授 ： 林俊清

高雄醫學大學/藥學院/天然藥物研究所碩士班/博士(2011年)

https://doi.org/10.6832/KMU.2011.00041

摘要

人類呼吸道融合病毒會引起嬰幼兒的下呼吸道感染，造成細支氣管炎和肺炎，目前臨床用藥有限和無疫苗可用，因此本研究探討中草藥防治 Respiratory syncytial virus (RSV)感染之可行性。
升麻葛根湯曾報導有抗麻疹病毒和腸病毒71型的感染，因此選擇了升麻葛根湯及其成份，利用病毒溶斑減少分析法，研究抗RSV病毒之活性成分和機轉，結果顯示：升麻葛根湯與其相關組成，升麻、甘草、生薑、芍藥和葛根皆具有抗RSV病毒活性。在A549細胞抑制RSV病毒感染50%的劑量(IC50)分別為升麻葛根湯：34.2 ± 1.2 μg/ml，升麻：29.4 ± 1.8 μg/ml，甘草：36.7 ± 2.2 μg/ml，生薑：49.2 ± 2.4 μg/ml，芍藥：78.6 ± 3.2 μg/ml，葛根：198.8 ± 8.6μg/ml；而在HEp-2細胞抑制RSV病毒感染50%的劑量(IC50)分別為升麻葛根湯：82.8 ± 2.6 μg/ml，升麻：42.1 ± 1.2 μg/ml，甘草：90.2 ± 5.1 μg/ml，生薑：112.5 ± 3.4 μg/ml，芍藥：92.3 ± 1.6 μg/ml，葛根：295.6 ± 9.8μg/ml，結果顯示升麻葛根湯與升麻都可以非常有效地抑制RSV感染。因此以升麻葛根湯和升麻進行時間效應實驗，發現這兩種藥物在病毒感染前後皆有抗病毒活性，表現出具有預防和治療之功效。抗病毒機轉實驗結果顯示，升麻葛根湯和升麻皆可以有效地抑制病毒的吸附和穿透，而且兩者都會刺激細胞產生干擾素，抑制RSV病毒的複製。此一研究結果強烈地暗示升麻葛根湯具有治療RSV病毒感染的效果，而升麻則可做為開發抗病毒藥物的來源。
以升麻純化合物升麻素(cimicifugin) 進行的抗病毒實驗顯示：cimicifugin在A549細胞的IC50為3.6± 0.8 μg/ml，SI值為69.4；在HEp-2細胞的IC50為29.3± 1.9 μg/ml，SI值為8.5。實驗結果展現出cimicifugin具有良好的抗病毒效果，而且是新發現。
時間效應實驗顯示cimicifugin在感染前後皆有抗病毒的活性；藥物預防RSV感染的作用機轉結果顯示：cimicifugin在A549與HEp-2細胞抑制病毒附著之IC50值分別為2.8 ± 0.3 μg/ml和29.3± 1.9 μg/ml；在抑制病毒穿透能力之IC50值則分別為13.6 ± 1.1 μg/ml∼33.1± 1.8 μg/ml和5.5± 0.6 μg/ml〜52.6 ± 2.7 μg/ml。

藥物治療RSV感染後的作用機轉結果顯示：cimicifugin會刺激細胞產生干擾素，抑制感染後RSV病毒的複製。
本研究結果顯示升麻葛根湯及升麻與cimicifugin，都有預防和治療RSV感染的潛力，未來可以再深入研究其抗病毒的作用機轉，而本研究結果也可以提供未來研發抗RSV病毒藥物的參考。

關鍵字

呼吸道融合病毒 ； 升麻葛根湯 ； 升麻素 ； 抗病毒機轉

並列摘要

Human respiratory syncytial virus (HRSV) is a major viral pathogen of lower respiratory tract infection in infants and children under 5 years of ages. Currently, no vaccines are available for prevention of HRSV infection. The use of antiviral drug, ribavirin was restricted on its efficacy, side effect, high cost, and difficulties with drug delivery. In this study, we wanted to find potential herbal candidate to manage HRSV infection.
Sheng-Ma-Ge-Gen-Tang (SMGGT) has been used against pediatric viral infection for thousands of year in ancient China. SMGGT has been found to be active against measles virus and enterovirus 71. Therefore, we tested the SMGGT and its related ingredients against HRSV by plaque reduction assay. The results showed that hot water crude extracts of SMGGT and its related components were all with the anti-RSV activities.The IC50 on A549 cells of the crude extracts of SMGGT, Cimicifuga foetida L., Glycyrrhiza uralensis Fischer et DC., Zingiber officinale Roscoe , Paeonia lactiflora Pallas, and Pueraria lobata Ohwi were 34.2 ± 1.2 μg/ml, 29.4 ± 1.8 μg/ml, 36.7 ± 2.2 μg/ml, 49.2 ± 2.4 μg/ml, 78.6 ± 3.2 μg/ml, and 198.8 ± 8.6μg/ml, respectively. The IC50 on HEp-2 cells of the crude extracts of SMGGT, C. foetida, G. uralensis, Z. officinale , P. lactiflora, and P. lobata were 82.8 ± 2.6 μg/ml, 42.1 ± 1.2 μg/ml, 90.2 ± 5.1 μg/ml, 112.5 ± 3.4 μg/ml, 92.3 ± 1.6 μg/ml, and 295.6 ± 9.8μg/ml, respectively. These results indicated that SMGGT and C. foetida were more effective against HRSV infection. The result of time course assay indicated that SMGGT and C. foetida exhibited their anti-HRSV activity both before and after HRSV infection. Therefore, they had both preventive and therapeutic potential. Study the antiviral mechanisms of SMGGT and C. foetida showed that they can both inhibit virus attachment and penetration to cells, and stimulated interferon-β to defense HRSV after viral inoculation. These data revealed SMGGT could be effective to manage HRSV infection in young children and C. foetida could be a sourse for developing antiviral drug.
Cimicifugin, a compound of C. foetida , was tested the ability of anti-RSV activity. The IC50 and SI of cimicifugin on A549 cells were 3.6± 0.8 μg/ml and 33.5；while on HEp-2 cells were 29.3± 1.9 μg/ml and 3.5. These results indicated this was a novel finding that cimicifugin could inhibit the plaque formation of HRSV in vitro.
In order to understand the anti-RSV mechanisms, we had time course assay, attachment assay, penetration assay and interferon assay. The results of time course assay showed that cimicifugin exhibited anti-HRSV activity before and after HRSV infection. Therefore, cimicifugin had preventive and therapeutic potential. The results of attachment assay of cimicifugin showed that the IC50 against HRSV attachment activity on A549 or HEp-2 cells were 2.8 ± 0.3 μg/ml or 29.3± 1.9 μg/ml. Its IC50 against HRSV penetration activity on A549 or HEp-2 cells after 20min~60min treatment ranged from 13.6 ± 1.1 μg/ml∼33.1± 1.8 μg/ml and 5.5± 0.6 μg/ml〜52.6 ± 2.7 μg/ml, respectively.
Cimicifugin could stimulate interferon production. Therefore, its anti-HRSV activity after viral infection was mediated by interferon.
Our results have proven that SMGGT, C. foetida and cimicifugin are effective to manage HRSV infection by inhibiting HRSV induced cytotoxicity. The mechanisms could be mediated by inhibiting viral attachment, suppressing viral penetration and by stimulating IFN-β. This study could be helpful to develop effective anti-HRSV agents.

並列關鍵字

Human respiratory syncytial virus ； antiviral mechanism ； Sheng-Ma-Ge-Gen-Tang ； cimicifugin

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