Evaluation of Bioactive Extractives from Heartwood of Taiwan Red Cypress(Chamaecyparis formosensis Matsum.)

摘 要 紅檜（Chamaecyparis formosensis Matsum.）為台灣特有之珍貴樹種，為了評估其心材抽出物之生物活性，本論文一方面除利用水蒸餾法萃取紅檜心材精油，並以氣相層析―質譜儀分析鑑定其組成及含量，同時利用抗病媒蚊幼蟲試驗及衣魚致死試驗，評估精油之生物活性。此外，亦利用甲醇萃取紅檜之心材抽出物，並進行各種生物活性之評估。試驗結果顯示，紅檜精油具有極佳的抗病媒蚊幼蟲能力，當濃度在100 μg/mL時，精油對埃及斑蚊幼蟲及白線斑蚊之致死率均達到100%；在抗衣魚活性方面，紅檜精油對衣魚具有顯著的毒殺作用，精油濃度0.16 mg/cm3，作用2 hr後，可達100%的毒殺效果。由海蝦致死試驗結果發現，紅檜抽出物具有相當大的潛力，可繼續進行腫瘤細胞毒性試驗。同時，紅檜心材抽出物具有良好的抗真菌活性，由抗真菌試驗結果得知，於100 g/mL的濃度下，抗菌活性依序為乙酸乙酯可溶部>甲醇抽出物>乙酸乙酯不可溶部。本研究續利用管柱層析與薄層層析等技術，對分離乙酸乙酯可溶部進行純化，於此分離部中分離鑑定出2個倍半萜類（T-Muurolol與5α,7αH-Eudesm-11(13)en- 4α-ol），2個二萜類（Isohinokiol及6,12-Dehydroxyabieta-5,8,11,13- tetraene-7-one）。至於抗氧化活性方面，紅檜心材成分並不具有顯著之抗氧化活性。綜合本研究所得之結果，紅檜之抽出成分具有多樣之生物活性，值得未來繼續針對其所含活性成分之確切結構及其作用機制作進一步的深入探討。Summary Taiwan red cypress (Chamaecyparis formosensis Matsum.) is an endemic precious tree specie grown in Taiwan. In this thesis, the bioactivities of extractives from Taiwan red cypress were investigated. Essential oil of Taiwan red cypress was prepared by water distillation and the chemical composition of essential oil was further analyzed by GC-MS. According to the results obtained from antimosquito and silverfish lethality assays, essential oil presented the good bioactivities against two kinds of organisms. It completely killed the larvae of A. aegypti and A. albopictus at the dosage of 100 μg/mL. As regard to silverfish lethality assay, we observed that the essential oil also had great empoisoned execution. While the concentration of essential oil dosage at 0.16 mg/cm3, the mortality was 100% after 2 hours treatment. In a parallel experiment, the wood chips were extracted with methanol. Its bioactivities were evaluated by using a number of bioassys, including brine shrimp test (BST), antioxidative activity assay and antifungal assay. In the meantime, the structures of the compounds, which isolated from total crude extract, were separated analyzed with chromatography as well as spectroscopy. The result of BST shown the extractives of Taiwan red cypress had the potential to further study their antitumor activity. The extractives also revealed significantly antifungal activity. The order of antifungal activities were EtOAc fraction >MeOH extract > EtOAc insolvable fraction. Following by chromatography separation and spectral analysis, four terpenoids were identified from active fraction, including T-Muurolol, 5α,7αH-eudesm-11(13)en-4α-ol, Isohinokiol and 6,12-De hydroxyabieta-5,8,11,13-tetraene-7-one. As regard to the antioxidative activity, the extractives of Taiwan red cypress heartwood did not possessed the good antioxidative activity so far. According to the results obtained from this study, the extractives of Taiwan red cypress possess multiple bioactivities. It is worthy to continue investigating its activity mechanisms and the exact structures of active components in the future.目 次 頁次 摘要 …………………………………………… i Summary ……………………………………… ii 目次………………………………………………… iv 表目次 ………………………………………… vii 圖目次 ………………………………………… ix 一、前言 ……………………………………… 1 二、文獻回顧 ………………………………… 4 (一) 精油的定義與萃取 …………………… 5 1. 蒸餾法 ……………………………………… 5 2. 同時水蒸氣蒸餾法 ………………………… 6 3. 脂法 ………………………………………… 7 4. 壓榨法 ……………………………………… 7 5. 超臨界萃取法 ……………………………… 8 6. 微波萃取法 ………………………………… 8 (二) 成分分析之研究方法 …………………… 9 1. 層析法 ……………………………………… 9 2. 核磁共振光譜分析 ………………………… 12 (三) 紅檜抽出物研究 ………………………… 14 (四) 與本論文相關之木材抽出物生物活性研究 27 1. 抗病媒蚊活性 ……………………………… 27 2. 抗衣魚活性 ………………………………… 28 3. 細胞毒性前篩選（Pre-screen）試驗 …… 29 4. 抗氧化活性與植物成分 …………………… 29 5. 木材之天然耐腐朽性 ……………………… 32 三、材料與方法 ……………………………… 38 (一) 試驗材料與成分萃取 …………………… 38 1. 試驗材料與儀器 …………………………… 38 (二) 成分萃取及分析 ………………………… 40 1. 精油萃取與成分分析 ……………………… 40 2. 抽出物之萃取與分離 ……………………… 41 3. 酚類化合物總量測定 ……………………… 43 (三) 生物活性試驗 …………………………… 44 1. 抗病媒蚊幼蟲試驗 ………………………… 44 2. 衣魚致死試驗 ……………………………… 45 3. 海蝦致死試驗 ……………………………… 46 4. 抗氧化活性試驗 …………………………… 47 5. 抗真菌活性試驗 …………………………… 48 四、結果與討論 ……………………………… 50 (一) 抽出物之含量及其組成分析 …………… 50 1. 精油收率、組成、含量及分析 …………… 50 2. 甲醇抽出物之收率 ………………………… 54 3. 甲醇抽出物之初步分離 …………………… 54 4. 酚類化合物總量測定 ……………………… 55 (二) 生物活性試驗 …………………………… 56 1. 抗病媒蚊幼蟲試驗 ………………………… 56 2. 衣魚致死試驗評估 ………………………… 59 3. 海蝦致死試驗評估 ………………………… 60 4. 抗氧化活性試驗 …………………………… 62 5. 抗真菌活性試驗 …………………………… 66 6. 成分分析及鑑定 …………………………… 68 五、結論 ……………………………………… 76 六、參考文獻 ………………………………… 7

紅檜表達序列標籤及其萜類生合成基因的選殖與功能表現

Chamaecyparis formosensis Matusm. is an endemic precious conifer grown in Taiwan. We used subtractive hybridization method to establish the expressed sequence tags (ESTs) in seedling and phloem of C. formosensis. Totally 384 and 452 ESTs were generated from seeding and phloem, individually. A blastx analyzed revealed that ESTs of seedling related to protein synthesis represented about 26%. The ESTs of phloem related to function of secondary metabolism and cell cycle and cell division represented about 18%. Morwover, to investigate the terpene synthesis mechanism in C. formosensis, full length of putative mono-TPS (Cf-Pin) and sesqui-TPS (Cf-Cad) were obtained by PCR method and RACE extension; and then we used recombinant protein from Escherichia coli were incubated individual with geranyl diphophate (GPP) and farnesyl diphosphate (FPP), which produced one major product, the structure of which was elucidated by gas chromatography/mass spectrometry (GC/MS) analysis. GC/MS analysis, Kovats index and MS matching with authentic standards revealed that the major products were α-pinene and β-cadinene. We cloned a mono-TPS (Cf-mono, full length: 1845 bp) and a di-TPS (Cf-di, full length: 1602 bp). We also cloned upstream of the terpene synthase including geranylgeranyl diphosphate synthase (full length of Cf-GGPPS has 1170 bp) and HMG-CoA reductase (partial sequence of Cf-HMGR has 1290 bp) of C. formosensis. To study analysis the volatile compounds of C. formosensis, in addition to the essential oil of leaves was prepared by hydrodistillation, the solid phase micro extraction technology was also conducted to collect the compounds emitted from leaves. Moreover, the leaves of C. formosensis were treated by methyl jasmonate (MeJA) to mimic it wounded or attacked by fungus or insects. The results in our study, totally 108 compounds were identified from C. formosensis, the amount of α-pinene is 30.29% is the most dominant compound in the volatile compounds emitted from leaves; however, β-myrcene (22.45%) is the most dominant compound in the essential oil. After MeJA treatment, the amount of volatile compound was increased to 1.92-fold. Meanwhile, we also found that the amount of volatiles would reach to the highest concentration after 5 hr treatment at the dosage of 10 mM MeJA.紅檜(Chamaecyparis formosensis Matsum.)為台灣特有樹種，本研究以大量片段的DNA序列分析策略，使用扣除雜合反應建構紅檜小苗以及成熟木材韌皮部表現差異的cDNA資料庫，結果分別獲得384及452筆基因表達序列標籤，藉此探討可能之蛋白質功能及其傳達之遺傳訊息。利用blantx分析結果發現，所獲得之小苗基因功能多與蛋白質合成有關，基因數目約佔全體的26%；韌皮部之基因功能則以二次代謝作用與細胞週期和細胞分裂相關者佔較多數，約佔總數的18%。另一方面，並針對紅檜的萜類合成酶(terpene synthase, TPS)進行研究，共獲得單萜合成酶(Cf-Pin, GenBank accession No. EU099434)與倍半萜合成酶(Cf-Cad, Genbank accession No. JN715077)兩個萜類合成酶，而為了進一步研究這些紅檜TPS的功能，將TPS以載體轉入大腸桿菌中大量表現重組蛋白質，經純化後Cf-Pin與Cf-Cad的重組蛋白質則分別利用前驅物GPP與FPP進行萜類合成酶的活性鑑定，所得之產物續利用氣相層析質譜分析確定其結構，經質譜分析結果及比對KI值確定重組蛋白質反應的產物為α-pinene及β-cadinene。此外，我們亦獲得一條推測為單萜合成酶(Cf-mono，基因全長為1845 bp)與一條雙萜合成酶(Cf-di，1602 bp)。同時針對萜類合成的相關合成酶也選殖到上游的geranylgeranyl diphosphate synthase (Cf-GGPPS全長基因1170 bp)與HMG-CoA reductase (Cf-HMGR片段基因1290 bp)。在揮發性成分的研究方面，本研究除利用水蒸餾法製備精油外，並利用固相微萃取技術收集葉子的揮發性成分，續以氣相層析質譜鑑定成分。此外，利用茉莉酸甲酯模擬葉子受到機械創傷或是昆蟲、真菌的侵害後，其揮發性成分的變化。結果共鑑定出108種化合物，其中以α-pinene為最主要成分，佔30.29%；而在精油中則是以β-myrcene為主成分，佔22.45%。紅檜葉子在茉莉酸甲酯處理1 hr後，其揮發性成分之釋出量大幅提高，揮發量為未以茉莉酸甲酯處理葉子的1.92倍；而在茉莉酸甲酯處理後之經時變化，10 mM茉莉酸甲酯處理5 hr後，其揮發量增加最多，之後揮發量就開始減少。目錄 頁次 摘要………………………………………………………………… Ⅰ Abstract……………………………………………………… Ⅲ 目錄………………………………………………………………… Ⅴ 表目錄……………………………………………………………… Ⅵ 圖目錄……………………………………………………………… Ⅶ 一、前言…………………………………………………………… 1 二、文獻回顧……………………………………………………… 4 (一)臺灣特有珍貴樹種 ─ 紅檜........ 4 (二)萜類化合物及其生合成路徑........ 6 (三)表達序列標籤(Expressed sequence tags, ESTs)13 (四)外在因子對萜類的影響.. 15 (五)紅檜的基因體研究.. 17 三、材料與方法…………………………………………………… 19 (一)紅檜基因體學研究 – ESTs的建立.......... 19 (二)紅檜萜類生合成基因之選殖及功能表現. 22 (三)茉莉酸甲酯處理對紅檜葉子揮發成分含量之影響.. 32 四、結果與討論…………………………………………………….. 35 (一)紅檜基因體學研究 – ESTs的建立........ 35 (二)紅檜萜類生合成基因之選殖及功能表現.... 41 (三)茉莉酸甲酯處理對紅檜葉子揮發成分含量之影響. 70 五、結論……………………………………………………………... 80 六、參考文獻………………………………………………………... 83 七、附錄…………………………………………………………….. 9

茉莉酸甲酯對紅檜葉子揮發成分含量之影響

紅檜為台灣特有樹種，木材具有極佳的耐久性並具特殊的香味。為瞭解紅檜葉子的揮發性成分組成，本研究除利用水蒸氣蒸餾法製備精油外，並利用固相微萃取技術收集葉子的揮發性成分，續以氣相層析質譜鑑定成分。此外，本研究並以茉莉酸甲酯模擬葉子受到機械創傷或是昆蟲、真菌的侵害後，其揮發性成分的變化。由分析結果可知，共108種紅檜葉子之化合物被鑑定出，在揮發性成分中以α-Pinene為最主要成分，佔30.29%，而在精油中則是以β-Myrcene為主成分，佔22.45%。紅檜葉子在茉莉酸甲酯處理後，其揮發性成分之釋出量大幅提高，揮發量為未以茉莉酸甲酯處理的葉子的1.92倍；而在茉莉酸甲酯處理後對於時間上的影響上，在施加10 mM茉莉酸甲酯經過5 hr後，其揮發量增加最多，之後揮發量就開始減少。Chamaecyparis formosensis is an endemic tree grown in Taiwan. The durability of C. formosensis wood is excellent and with fragrance odor. In order to analysis the volatile compounds of C. formosensis, in addition to the essential oil of leaves was prepared by water distillation, the solid phase micro extraction technology was also conducted to collect the compounds emitted from leaves. Moreover, the leaves of C. formosensis were treated by methyl jasmonate (MeJA) to mimic it wounded or attacked by fungus or insects. According to the results obtained in our study, totally 108 compounds were identified from C. formosensis, the amount of α-Pinene is 30.29% is the most dominant compound in the volatile compound emitted from leaves; however, β-Myrcene (22.45%) is the most dominant compound in the essential oil. After MeJA treatment, the amount of volatile compound was increased to 1.92-fold. Meanwhile, we also found that the amount of volatiles would reach to the highest concentration after 5 hr treatment at the dosage of 10 mM MeJA