臺灣博碩士論文加值系統

人類呼吸道融合病毒（Human respiratory syncytial virus，HRSV）是造成嬰幼兒下呼吸道病毒感染的首要病毒，同時也是導致老年人死於病毒感染的第二順位致病原，由於目前的臨床用藥只有Ribavirin，但是它藥效不佳又有毒性，另外也沒有疫苗可用，因此本論文研究中草藥防治HRSV感染的可行性。
小青龍湯是中國傳統醫學常用的複方，它被用於治療包括肺炎、鼻炎、感冒、流行性感冒、支氣管炎、氣喘及百日咳等呼吸系統疾病。過去研究顯示小青龍湯能抑制流行性感冒病毒和人類巨細胞病毒的感染，因此選擇小青龍湯及其組成單方，利用病毒溶斑減少法，研究抗HRSV之活性成分及機轉。結果顯示：小青龍湯及其組成之桂枝、麻黃、芍藥、五味子和炙甘草都具有抗HRSV的活性，抑制病毒感染50％的劑量（IC50）分別為54.9 ± 4.8 μg/ml、7.2 ± 0.3 μg/ml、14.9 ± 1.8 μg/ml、60.1 ± 2.3 μg/ml、150.9 ± 13.6 μg/ml和176.9 ± 9.7 μg/ml。根據IC50及選擇指數值（SI值）的結果顯示小青龍湯和桂枝都可以有效且安全地抑制HRSV感染。時間效應實驗發現小青龍湯及桂枝在感染前後皆有抗HRSV的活性，表示兼具預防和治療之功效。抗病毒機轉結果顯示：小青龍湯和桂枝都可以有效的抑制HRSV的吸附及穿透作用。小青龍湯必須在高濃度（300 μg/ml）時才能抑制HRSV所誘導的細胞融合反應，而桂枝在300 μg/ml時則是完全抑制。小青龍湯只在3 μg/ml及桂枝在3 μg/ml和10 μg/ml能刺激未感染病毒的細胞分泌干擾素；小青龍湯在3 μg/ml~300 μg/ml會降低感染病毒的細胞分泌干擾素，桂枝在10 μg/ml~300 μg/ml能刺激被感染細胞分泌干擾素。小青龍湯和桂枝都可以抑制被HRSV感染的細胞分泌會引起發炎的腫瘤壞死因子。
桂枝相關21種純化合物：cinnamaldehyde、trans-cinnamic acid、cinnamyl acetate、benzaldehyde、cuminaldehyde、vanillin、o-anisaldehyde、2-methoxycinnamic acid、2-methoxycinnamaldehyde、cinnamyl alcohol、hydrocinnamaldehyde、methyl salicylate、methyl salicylaldehyde、salicylaldehyde、salicylic acid、catechin、epicatechin、protocatechuic acid、limonene、linalool和coumarin及五味子與芍藥都含有的γ-linolenic acid成分，當中只有cinnamaldehyde、2-methoxycinnamaldehyde、limonene和γ-linolenic acid有較強的抗HRSV活性，IC50分別為5.6 ± 0.2 μg/ml、6.3 ± 0.3 μg/ml、9.1 ± 0.6 μg/ml和9.4 ± 0.9 μg/ml。時間效應結果顯示cinnamaldehyde在感染前後皆有抗HRSV的活性。藥物預防HRSV感染的作用機轉結果顯示：cinnamaldehyde預防HRSV吸附之IC50值為3.3 ± 0.2 μg/ml；抑制穿透之IC50值為3.4 ± 0.3 μg/ml～5.1 ± 0.5 μg/ml。藥物抑制HRSV感染後繁殖病毒的作用機轉結果顯示：cinnamaldehyde在0.25 μg/ml~8 μg/ml能刺激未感染的細胞產生干擾素，但只在0.25 μg/ml能刺激被HRSV感染的細胞分泌干擾素。Cinnamaldehyde在0.25 μg/ml~8 μg/ml能抑制被HRSV感染的細胞分泌會引起發炎的腫瘤壞死因子。另外，cinnamaldehyde會抑制HRSV所誘導的細胞融合反應。
本論文研究結果證明小青龍湯、桂枝及cinnamaldehyde都有預防和治療HRSV感染的潛力，未來可再深入探討其抑制感染後的HRSV複製及藥物的應用性。

Human respiratory syncytial virus (HRSV) is a significant etiology of lower respiratory tract infection in infants and children under 5 years of ages. HRSV is also the second leading cause of viral death in elderly individuals. Currently, there is no vaccine available for prevention of HRSV infection. The only antiviral agent approved for treatment of RSV disease is ribavirin, but due to efficacy, toxicity and high cost, it has only limited uses. In this study, we wanted to find potential herbal candidates to manage HRSV infection.
Xiao-Qing-Long-Tang (XQLT) is a traditional Chinese medicine formula, which had been used to treat the respiratory tract diseases, including pneumonia, rhinitis, colds, influenza, bronchitis, wheeze and pertussis. XQLT has been reported to inhibit influenza virus and human cytomegalovirus and its related herbal ingredients have antiviral activity. Therefore, we tested the XQLT and its related ingredients against HRSV by the plaque reduction assay. The results showed that hot water crude extracts of XQLT and its ingredients, namely, Ephedra sinica Stapf., Cinnamomum cassia Blume, Paeonia lactiflora Pallas, Schizandra chinensis Baill. and Glycyrrhiza uralensis Fisch., were effective against HRSV-induced plaque formation. The IC50 of the above against HRSV infection were 54.9 ± 4.8 μg/ml, 7.2 ± 0.3 μg/ml, 14.9 ± 1.8 μg/ml, 60.1 ± 2.3 μg/ml, 150.9 ± 13.6 μg/ml and 176.9 ± 9.7 μg/ml, respectively. The IC50 and selectivity index indicated that XQLT and C. cassia were effective and safe against HRSV infection.
The results of time course assays indicated that XQLT and C. cassia exhibited anti-HRSV activity before and after HRSV infection. Therefore, they had both preventive and therapeutic potential. Study on the antiviral mechanisms of XQLT and C. cassia showed that they can inhibit HRSV attachment and penetration to cells. XQLT and C. cassia can inhibit HRSV-induced syncytial formation on 300 μg/ml. Treated cells without HRSV infection with XQLT 3 μg/ml or C. cassia 3 μg/ml or 10 μg/ml can stimulate interferon-β（IFN-β）secretion. Treated with XQLT 3 μg/ml~300 μg/ml can reduce IFN-β secretion after HRSV infection. Treated with C. cassia 10 μg/ml～300 μg/ml can stimulate IFN-β secretion after HRSV infection. XQLT and C. cassia both can inhibit proinflammatory cytokine, tumor necrosis factor-α（TNF-α）secretion after HRSV infection.
Furthermore, we study activity of pure compounds of C. cassia, namely cinnamaldehyde, trans-cinnamic acid, cinnamyl acetate, benzaldehyde, cuminaldehyde, vanillin, o-anisaldehyde, 2-methoxycinnamic acid, 2-methoxycinnamaldehyde, cinnamyl alcohol, hydrocinnamaldehyde, methyl salicylate, methyl salicylaldehyde, salicylaldehyde, salicylic acid, catechin, epicatechin, protocatechuic acid, limonene, linalool and coumarin. The pure compound γ-linolenic acid of S. chinensis and P. lactiflora was evaluated. Only cinnamaldehyde, 2-methoxycinnamaldehyde, limonene and γ-linolenic acid were effective against HRSV-induced plaque formation. The IC50 of the above against HRSV infection were 5.6 ± 0.2 μg/ml, 6.3 ± 0.3 μg/ml, 9.1 ± 0.6 μg/ml and 9.4 ± 0.9 μg/ml, respectively. The results of time course assays showed that cinnamaldehyde exhibited anti-HRSV activity before and after HRSV infection. Therefore, cinnamaldehyde had preventive and therapeutic potential. The assay result of cinnamaldehyde showed that the IC50 value against HRSV attachment activity was 3.3 ± 0.2 μg/ml. Its IC50 values against HRSV penetration activity after 20 min~60 min treatment ranged from 3.4 ± 0.3 μg/ml～5.1 ± 0.5 μg/ml. Cinnamaldehyde can inhibit HRSV-induced syncytial formation. Treated with cinnamaldehyde 0.25 μg/ml~8 μg/ml before or 0.25 μg/ml after HRSV infection can stimulate IFN-β secretion. Cinnamaldehyde can inhibit proinflammatory cytokine, tumor necrosis factor-α（TNF-α）secretion after HRSV infection.
Our results proved that XQLT, C. cassia and cinnamaldehyde are effective to manage HRSV infection by inhibiting HRSV induced cytotoxicity. The mechanisms could be mediated by inhibiting viral attachment, penetration and HRSV-induced syncytial formation. This study may be helpful to develop effective anti-HRSV agents.

中文摘要--------------------------------------------1
英文摘要--------------------------------------------4
壹、前言
（一）人類呼吸道融合病毒介紹------------------------6
（二）小青龍湯及其相關成分之介紹--------------------13
（三）本論文所選用的桂枝純化合物之生物活性介紹------61
（四）γ-linolenic acid（五味子及芍藥所含之成分）：生物活性--81
貳、材料與方法--------------------------------------83
參、結果--------------------------------------------100
肆、討論--------------------------------------------136
伍、總結論------------------------------------------153
陸、圖表--------------------------------------------157
柒、參考文獻----------------------------------------198
捌、學會發表----------------------------------------237

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