臺灣博碩士論文加值系統

腸病毒與腺病毒兩者皆屬腸道病毒，為台灣地區常見的急性傳染病。目前對於這兩種病毒屬並沒有專屬性強且有效的化學藥物，由於感染腸道病毒後的症狀，在中醫學觀點來判斷，皆屬腸道濕熱所引起的發熱，在中醫上主要投與清熱解毒藥物做治療。中草藥植物是病毒治療新藥希望來源，因此本實驗將針對十種常見清熱解毒之中草藥（懸鉤子、骨碎補、山豆根、苦蘵、忍冬、南國小薊、三葉五加、蟛蜞菊、小毛氈苔、射干）及另外四種透過誘變劑誘變之射干不定根（T05、T1、Ori、Col）進行抗腸病毒（EV71）與腺病毒（ADV41）的活性分析。
本研究以MTT assay建立中草藥抗病毒活性快速大量篩選體外模式，初步抗病毒活性篩選結果，射干誘變株T05、T1之萃取物有明顯的抗ADV41病毒活性，篩選指標SI值為139、59.7；而射干誘變株Ori、T05萃取物則有明顯抗EV71病毒活性，篩選指標SI值為274.5、42.2。進一步選擇具明顯抗病毒活性（高SI值）的中草藥以分時添加測試可能藥理特性，推測射干誘變株T1對ADV41初期感染具有抑制病毒複製之活性，有效劑量285及1140 μg/mL作用下分別為52及42％病毒活性抑制比率；而射干誘變株T05及Ori對EV71具有明顯之細胞保護作用，有效劑量1200 μg/mL作用下分別為77及52％病毒活性抑制比率。
此外，比較三種化學誘變劑包括colchicines（秋水仙鹼）、oryzalin（氨磺靈）及trifluralin（氟樂靈）對於射干不定根抗病毒之研究。射干透過植物組織培養方式，經由誘變劑誘變出具有較高類黃酮含量之品系，其中發現射干誘變株T05及T1分別對EV71及ADV41具有良好抗病毒效果。
中草藥抗病毒活性的大量篩選模式及藥理分析測試，可有效找尋具抗腸病毒及腺病毒活性的中草藥及潛力化合物質，配合誘變劑可提高有
效成分之產量及質量，可作為未來研發病毒治療藥物的基礎。

Enterovirus and Adenovirus both are among the intestinal virus for common acute infectious disease in Taiwan. There is no highly specific and effective chemical drugs for these two virus, and the symptoms of infection was belong to the fever caused by intestinal heat, in Chinese medicine is mainly used to make the drug detoxification treatment, Chinese herbal medicine is the hope of development of new drug for intestinal virus.
We established a high throughput screening system of anti-viral activity by MTT assay in vitro, and tested 10 kinds of traditional Chinese herb extracts and 4 kinds of Belamcanda chinensis mutagen extracts. Preliminary antiviral activity screening result showed that the extract of Belamcanda chinensis mutagenic strains T05 and T1 has significant anti-adenovirus type 41(ADV41) activity, with the selectivity index(SI) value of 139, 59.7; and the extracts of Belamcanda chinensis mutagenic strains Ori and T05 has significant anti-enterovirus type 71(EV71) activity, with the SI value of 274.5, 42.2 respectively. Further, we chose Chinese herb extracts with significant antiviral activity (high SI values) to analyze the possible pharmacological properties by time course test. The results showed that Belamcanda chinensis mutagenic strains T1 extracts was found to inhibit the replication of ADV41 during early infection, the inhibition rate were 52% and 42% with the effective dose of 285 and 1140 μg/mL, respectively. The Belamcanda chinensis mutagenic strains T05 and Ori extract had cytoprotective effect against EV71, the inhibition rate were 77% and 52% with the effective dose of 1200 μg/mL, respectively.
Comparison of three chemical mutagens for anti-virus research Belamcanda chinensis adventitious roots include colchicines, oryzalin and trifluralin. Belamcanda chinensis mutagen by trifluralin has a high flavored content of strains via plant tissue culture.
High throughput Chinese herb screening system of anti-viral activity and analysis of the possible pharmacological properties with mutagenic agents may increase production and quality of the active ingredient can be used as the basis for the development of the new drug for the enterovirus

Abstract i
摘要 iii
目錄 v
圖目錄 viii
表目錄 ix
第一章 緒論 1
1.1 研究動機 1
1.2 文獻回顧 3
1.2.1 腸病毒簡介 3
1.2.2 腸病毒感染症狀及流行病學流行病學 3
1.2.3 腸病毒構造及感染性 4
1.2.4 腸病毒生活史 5
1.3 腺病毒簡介 9
1.3.1 腺病毒感染症及流行病學 9
1.3.2 腺病毒構造及感染性 10
1.3.3 腺病毒生活史 13
1.4病毒感染之中醫觀點與新藥開發 16
1.4.1 腸病毒感染症與中醫觀點 16
1.4.2 腺病毒感染症與中醫觀點 17
1.4.3 清熱解毒之中藥介紹 17
1.4.4 傳統中草藥與新藥開發 18
1.4.5 藥用植物透過人工誘變之應用 21
1.4.5.1 人工誘變技術在藥用植物中的未來展望 22
1.4.6 射干簡介 22
1.4.6.1 射干藥理作用 22
1.4.6.2 射干抗氧化和清除自由基的作用 24
1.4.6.3 射干抗癌作用 25
1.4.6.4 蛋白質激酶C 25
1.4.6.5 射干與蛋白激酶C活化作用 26
1.4.6.6 射干誘變株對黃酮生產之影響 26
1.5 抗病毒活性篩選方法的選擇 29
第二章 研究目的與架構 30
2.1 研究目的 30
2.2 實驗架構 30
第三章 材料與方法 32
3.1 細胞株 32
3.2 病毒株 32
3.3 半細胞培養致死(TCID50) 32
3.4 中草藥抗病毒活性篩選 33
3.4.1 藥物細胞毒性測試實驗 33
3.4.2 藥物抗病毒活性測試 34
3.4.2.1 MTT assay 34
3.4.3 藥理特性分析 34
第四章 結果 37
4.1 中草藥抗病毒活性之體外細胞篩選模式 37
4.1.1 中草藥細胞毒性測試 37
4.1.2 中草藥抗病毒活性篩選結果 39
4.1.3 抗病毒活性高SI值的中草藥之藥理特性分析 41
4.2 射干三種誘變劑之抗病毒效果比較 59
第五章 討論 61
5.1 中草藥抗病毒篩選結果之探討 61
5.2 植物組織培養透過化學誘變劑後之影響 63
5.2.1 化學誘變劑colchicine、oryzalin及trifluralin 63
5.2.2 射干誘變株有效抗病毒之化學成分 63
第六章 結論 65
參考文獻 66

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