Mosquitocidal Activities of Leaf Oil and Its Constituents from Calocedrus macrolepis var. formosana

埃及斑蚊與白線斑蚊是登革熱的主要病媒蚊，目前並無有效的登革熱疫苗，因此病媒蚊的防治是非常重要的措施。近年來許多專家學投入天然植物抽出物的研究，希望能找出有效的抗病媒蚊成分來取代化學藥劑的使用。台灣肖楠(Calocedrus macrolepis var. formosana)為台灣特有的樹種，為了評估台灣肖楠葉部精油之抗病媒蚊活性，本試驗萃取了12種台灣肖楠葉子的精油，經由氣相層析-質譜儀(GC-MS)與群團分析，可依據精油成分將其分成3種化學品系:Limonene(檸檬烯)型、α-Pinene(α-蒎烯)型與混合型，皆具有不錯的抗病媒蚊活性，進一步評估精油成分的活性，其抗病媒蚊活性大小為(-)-Limonene > (+)-Limonene ≒ β-Caryophyllene ≒ (-)-α-Pinene >β-Myrcene ＞β-Pinene ≒ (+)-α-Pinene，以Limonene之抗病媒蚊活性最佳。 比較精油及其成分的抗病媒蚊試驗結果，顯示精油及其成分抗埃及斑蚊的活性比白線斑蚊較佳。本試驗亦探討化合物旋光性與抗病媒蚊活性之間的關係，試驗結果顯示旋光性的不同會影響抗病媒蚊活性，而右旋與左旋對於抗病媒蚊活性之優劣則因化合物而異，台灣肖楠葉部精油中的Limonene與α-Pinene均以左旋結構的抗病媒蚊活性較強，(-)-Limonene對埃及斑蚊幼蟲之LC50僅為14μg/mL。進一步評估Limonene對白線斑蚊與埃及斑蚊幼蟲生長及成蚊之不孕性的影響，試驗結果發現在低濃度(10μg/mL)時，Limonene即可使病媒蚊幼蟲的化蛹率及羽化率下降，同時可以有效降低成蚊的產卵數以及卵的孵化率。綜合試驗結果顯示台灣肖楠葉部精油以及精油成分具有非常良好的抗病媒蚊潛能，值得進一步的開發與利用。Aedes aegypti and Aedes albopictus are main vectors of dengue fever. There is no effective vaccine for dengue fever thus far. Therefore, control of mosquito population is the best way to control and prevent dengue fever. Many experts have tried to find mosquitocidal natural products to replace synthetic insecticides in recent years. Calocedrus macrolepis var. formosana is an endemic tree species in Taiwan. To evaluate mosquitocidal activities of leaf oils from Calocedrus macrolepis var. formosana, leaf oils from twelve sources were extracted and chemical constituents were analyzed by GC-MS in this study. According to the chemical composition of essential oil, leaf oils of C. macrolepis var. formosana from different sources could be classified into three chemotypes: Limonene chemotype, α-Pinene chemotype and mixed chemotypes by cluster analyses. All chemotype oils possess the larvicidal activity against mosquito larvae. Larvicidal activities of main constituents of leaf oil were also evaluated, results ranked in descending order of larvicidal activity: (-)-Limonene > (+)-Limonene ≒ β-Caryophyllene ≒ (-)-α-Pinene > β-Myrcene ＞β-Pinene ≒ (+)-α-Pinene. Limonene possesses the best mosquitocidal activity. Results of larvicidal tests demonstrated that leaf oils from C. macrolepis var. formosana and its constitunets had better mosquitocidal activities against Ae. aegypti than Ae. albopictus. In addition, influence of the optical activity on the larvaicidal activity of enantiomers was investigated. Results indicated that the optical activity can affect larvicidal activities of enantiomers. Varied effects of the optical activity on larvicidal activities of enantiomers occurred in different constituents. Levorotatory(-) isomer of both Limonene and α-Pinene, main larvicidal constituents of leaf oil, possess the better larvicidal activity than that of dextrorotatory(+) isomer. (-)-Limonene had the best mosquito larvicidal activity with an LC50 of 14 μg/mL. Furthermore, the effects of Limonene on growth inhibiting, ovicidal activities and egg hatchability of Ae. aegypti and Ae. albopictus were also discussed in the research. Results showed the growth, pupal development and eclosion of mosquito pupae were retarded by Limonene at low concentration (10μg/mL). Limonene also interfered with oviposition and egg hatchability of mosquitoes. The average number of eggs laid (per female) and hatching rate of egg of mosquitos were significantly decreased after Limonene treatment. These data indicate that leaf oil of C. macrolepis var. formosana and its constituents have great potential as natural insecticide in mosquito control and are worth further exploring.目錄 表目次 .............................................. Ⅳ 圖目次 ............................................... Ⅵ 摘要 ................................................. Ⅹ Summary .............................................. XⅡ 壹、緒言 ............................................. 1 貳、文獻回顧 ......................................... 4 一、台灣肖楠抽出成分之研究 ........................... 4 二、植物天然物之抗病媒蚊活性 ......................... 5 (一)、蚊媒病與病媒蚊的防治 ........................... 5 1.蚊蟲的之生態 ...................................... 5 2.蚊蟲傳播的疾病 .................................... 6 3.病媒蚊的防治 ...................................... 7 (二)、植物抽出物的殺蚊幼蟲活性與生長抑制活性 ......... 9 1.植物抽出物的殺蚊幼蟲活性 .......................... 9 2.抽出成分的殺蚊幼蟲活性 ............................ 11 (1).碳氫化合物 ..................................... 12 (2).萜類化合物 ..................................... 13 a.單萜類........................................... 13 b.倍半萜類......................................... 15 c.二萜類........................................... 15 d.三萜類........................................... 15 (3).芳香族化合物 ................................... 16 (4).木酚素化合物.................................... 17 (5).生物鹼化合物.................................... 17 3.植物抽出物對蚊蟲的生長抑制活性 .................... 20 (三)、具蚊蟲驅避活性的植物抽出物 ..................... 22 (四)、具昆蟲費洛蒙功效之植物抽出成分 ................. 23 參、材料與方法 ....................................... 25 一、試驗材料 ......................................... 25 (一)、台灣肖楠 ....................................... 25 (二)、精油成分 ....................................... 26 二、試驗方法 ......................................... 26 (一)、精油之萃取 ..................................... 26 (二)、精油成分之鑑定 ................................. 26 (三)、化學品系分析 ................................... 27 (四)、抗蚊試驗方法 ................................... 27 1.蚊蟲飼養 .......................................... 27 2.抗病媒蚊幼蟲試驗 .................................. 28 3.精油成分對病媒蚊的生長抑制試驗 .................... 28 4.成蚊之不孕性影響試驗 .............................. 29 a.母蚊產卵率之計算 ................................. 29 b.孵化率之計算 ..................................... 30 (五)、統計分析........................................ 30 肆、結果與討論 ....................................... 31 一、台灣肖楠葉部精油之化學品系分析 ................... 31 (一)、精油收率 ....................................... 31 (二)、精油成分之分析與鑑定 ........................... 32 (三)、台灣肖楠葉部精油之化學品系分析.................. 38 (四)、栽植地對葉部精油成分之影響...................... 40 二、台灣肖楠葉部精油之抗病媒蚊活性 ................... 42 三、台灣肖楠葉部精油成分之抗病媒蚊活性 ............... 47 (一)、葉部精油主成分之抗病媒蚊活性探討................ 47 (二)、鏡相異構物之抗病媒蚊活性探討 ................... 50 四、Limonene對孑孓生長及成蚊不孕性的影響.............. 52 (一)、Limonene對孑孓生長狀況之影響.................... 52 (二)、Limonene對病媒蚊幼蟲羽化之影響.................. 59 (三)、Limonene對成蚊不孕性之影響...................... 62 伍、結論 ............................................. 68 陸、參考文獻 ......................................... 70 表目次 表1. 常見化學殺蟲劑之成效 Table 1.Effects of common chemical insecticides ...... 9 表2. 植物抽出物之殺蚊幼蟲活性 Table 2.Larvicidal activities of plant extracts ...... 10 表3. 異丁胺鹼類化合物的殺蚊幼蟲活性 Table 3.Larvicidal activities of isobutylamide alkaloids......... 18 表4. 薄荷精油和印度黃檀精油對蚊蟲的生長抑制活性 Table 4. Growth inhibiting activities of peppermint oil and Dalbergia sissoo oil against the larvae of Aedes aegypti, Culex pipiens and Anopheles stephensi ......................... 21 表5. 三種精油的驅蚊效果 Table 5.Repellent activities of Dalbergia sissoo oil, peppermint oil and mylol oil ........................ 23 表6. 成蚊之不孕性試驗之配對組別 Table 6. Treatments of adult mosquitos in sterile test.............. 30 表7. 台灣肖楠葉部精油的精油收率 Table 7.Yields of leaf oils from Calocedrus macrolepis var. formosana........................................ 31 表8. 台灣肖楠葉部精油之成分 Table 8.Constituents of leaf oils from Calocedrus macrolepis var. formosana ........................... 33 表 9. 不同來源之台灣肖楠葉部精油成分組成(Ⅰ) Table 9.Constituents of Calocedrus macrolepis var. formosana leaf oils from different sources (Ⅰ) ...... 34 表 10. 不同來源之台灣肖楠葉部精油成分組成(Ⅱ) Table 10.Constituents of Calocedrus macrolepis var. formosana leaf oils from different sources (Ⅱ) ...... 35 表11. 台灣肖楠葉部精油之單萜類及其含氧化合物、倍半萜類及其含氧化合物之含量 Table 11.Contents of terpenoids from Calocedrus macrolepis var. formosana leaf oils.................. 37 表12. 不同栽植地台灣肖楠葉子精油的主要成分含量 Table 12. Contents of main components of Calocedrus macrolepis var. formosana leaf oils from different sources.................... 41 表13. 台灣肖楠葉部(GH)精油對病媒蚊幼蟲之有效致死濃度 Table 13. Lethal concentrations (LC) of Calocedrus macrolepis var. formosana leaf oil (GH) against mosquito larvae............ 43 表14 精油成分對病媒蚊幼蟲之有效致死濃度 Table 14. Lethal concentrations (LC) of effective components from Calocedrus macrolepis var. formosana leaf oil against mosquito larvae .......................... 49 圖目次 圖1. 具抗病媒蚊活性之碳氫化合物 Fig.1.Chemical structures of hydrocarbons possessing mosquitocidal activities ............................. 12 圖2. 具抗病媒蚊活性之萜類化合物 Fig.2.Chemical structures of terpenoids possessing mosquitocidal activities ............................ 14 圖3. 具抗病媒蚊活性之芳香族化合物 Fig.3.Chemical structures of aromatic compounds possessing mosquitocidal activities ............................. 17 圖4. 具抗病媒蚊活性之生物鹼化合物 Fig.4.Chemical structures of alkaloids possessing mosquitocidal activities.............................. 19 圖5. K.scoparia種子油成分與產卵性費洛蒙化合物(5R,6S)-6-acetoxy-5- hexadecanolide Fig.5.Compounds from K. scoparia seed oil and oviposition pheromone (5R,6S)-6-acetoxy-5-hexadecanolide ......... 24 圖6. 台灣肖楠葉部精油之氣相層析圖 Fig.6.GC spectrum of leaf oil from Calocedrus macrolepis var. formosana....................................... 32 圖7. 台灣肖楠葉部精油成分中主要萜類化合物之化學結構式 Fig.7.Structures of main terpenoids from Calocedrus macrolepis var. formosana leaf oil .................. 36 圖8. 12種來源台灣肖楠精油成分含量之群團分析樹狀圖 Fig.8.Dendrogram obtained by cluster analysis of the percentage composition of essential oils from twelve sources of Calocedrus macrolepis var formosana ....... 39 圖9. 台灣肖楠葉部精油(GH)於不同濃度下對埃及斑蚊與白線斑蚊第4齡幼蟲的致死率 Fig.9. Mosquitocidal activities of Calocedrus macrolepis var. formosana (GH) against fourth instar larvae of Aedes aegypti and Aedes albopictus................. 43 圖10. 不同來源台灣肖楠葉部精油之抗埃及斑蚊4齡幼蟲活性 Fig.10.Larvicidal activities of Calocedrus macrolepis var. formosana leaf oils from different sources against fourth instar larvae of Aedes aegypti ..................45 圖11. α-Pinene與Limonene含量對精油抗病媒活性之影響 Fig.11. Effects of contents of α-Pinene and Limonene on the mosquitocidal activities of essential oils........ 46 圖12. 精油主成分(100μg/mL)之抗埃及斑蚊4齡幼蟲活性 Fig.12. Mosquitocidal activities of main components (100μg/mL) against fourth instar larvae of Aedes aegypti... 47 圖13. 精油主成分(100μg/mL)之抗白線斑蚊4齡幼蟲活性 Fig.13.Mosquitocidal activities of main components (100μg/mL) against fourth instar larvae of Aedes albopictus ....... 48 圖14. 具抗病媒蚊效果之鏡相異構物 Fig.14.Structures of enantiomers possessing mosquitocidal activities .......................................... 51 圖15. 鏡相異構物之對埃及斑蚊4齡幼蟲之抗蚊活性 Fig.15.Mosquitocidal activities of enantiomers against fourth instar larvae of Aedes aegypti ................ 51 圖16. 埃及斑蚊4齡幼蟲控制組生長狀況 Fig.16. Life history of fourth instar larvae of Aedes aegypti (control).................................... 53 圖17.埃及斑蚊4齡幼蟲以(+)-limonene(10μg/mL)處理後之生長狀況 Fig.17.Life history of fourth instar larvae of Aedes aegypti exposed to (+)-Limonene (10μg/mL) solution............................ 54 圖18.埃及斑蚊4齡幼蟲以(-)-limonene(10μg/mL)處理後之生長狀況 Fig.18.Life history of fourth instar larvae of Aedes aegypti exposed to (-)-Limonene (10μg/mL) solution....................... 55 圖19.白線斑蚊4齡幼蟲控制組生長狀況 Fig.19. Life history of fourth instar larvae of Aedes albopictus (control)................................. 56 圖20.白線斑蚊4齡幼蟲以(+)-Limonene(10μg/mL)處理後之生長狀況 Fig.20.Life history of fourth instar larvae of Aedes albopictus exposed to (+)-Limonene (10μg/mL) solution................ 57 圖21.白線斑蚊4齡幼蟲以(-)-Limonene(10μg/mL)處理後之生長狀況 Fig.21.Life history of fourth instar larvae of Aedes albopictus exposed to (-)-Limonene (10μg/mL) solution................. 58 圖22. Limonene對埃及斑蚊幼蟲羽化的影響 Fig.22. Effect of Limonene on the eclosion of Aedes aegypti larvae....................................... 60 圖23. Limonene對白線斑蚊幼蟲羽化的影響 Fig.23. Effect of Limonene on the eclosion of Aedes albopictus larvae..................................... 61 圖24. Limonene對埃及斑蚊產卵數的影響 Fig.24. Average of eggs laid (per female) of Aedes aegypti exposed to Limonene solution.......................... 63 圖25. Limonene對埃及斑蚊孵化率的影響 Fig.25. Egg hatchability of Aedes aegypti exposed to Limonene solution..................................... 64 圖26. Limonene對白線斑蚊產卵數的影響 Fig.26. Average of eggs laid (per female) of Aedes albopictus exposed to Limonene solution............... 65 圖27. Limonene對白線斑蚊卵孵化率的影響 Fig.27. Egg hatchability Aedes albopictus exposed to Limonene solution.................................... 6