柳杉抗真菌及抗蟲成分之分析與鑑定

Analyses and Identification of Antifungal and Insecticidal Compounds from Cryptomeria japonica

鄭森松(Sen-Sung Cheng)

指導教授 ： 張上鎮

國立臺灣大學/生物資源暨農學院/森林環境暨資源學研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.01815

摘要

柳杉（Cryptomeria japonica D. Don, Japanese cedar）為台灣重要造林樹種之一，如何利用此一資源已成為當前林業經營與林產利用的重要課題。因此，本研究分別以水和甲醇萃取柳杉各部位精油及抽出成分，進行生物活性之篩選，嘗試分離及純化柳杉中具抗木材腐朽菌、抗植物病原菌、抗白蟻和抗病媒蚊幼蟲等生物活性之主要成分。所得之純化合物則利用質譜、紅外線光譜及核磁共振光譜等儀器分析鑑定其化學結構，同時亦將純化合物進行各項試驗以確認其生物活性；另外，亦探討抽出成分結構與生物活性之相關性。

由氣相層析儀和氣相層析-質譜儀分析結果得知，心材精油主成分為γ-Cadinene（14.46%）、epi-Bicyclosesquiphellandrene（13.15%）及δ-Cadinene（12.86%）；邊材精油主成分為Isopimarol（23.11%）、Ferruginol（14.74%）；樹皮精油主成分為Ferruginol（16.81%）和δ-Cadinene（6.76%）；葉子精油主成分為ent-Kaur-16-ene（24.90%）和β-Elemol（18.84%）。又本研究由柳杉心材、邊材、葉子和樹皮中共分離出24種化合物，包括Bisabola-7(14),10- dien-1α,4α-diol、Khusinodiol、δ-Cadinol、α-Cadinol、γ-Eudesmol、β-Eudesmol、β-Elemol、Cryptomeridol、Ferruginol、Cryptojaponol、Isopimaric acid、7β-Hydroxydeoxocryptojaponol、Isopimarol、Isopimarinol、Phyllocladan-16α-ol、Sandaracopimaric acid、12-Hydroxy-6,7-secoabieta-8,11,13-trien-6,7-dial、ent-Kaur-16-ene、Sugiol、Cryptoresinol、2-Methylanthraquinone、6β,22- Dihydroxystigmasterol、(22S)-5α-Ergostane-3α,22-diol及β-Sitosterol。其中，Khusinodiol、2-Methylanthraquinone、6β,22-Dihydroxy-stigmasterol和(22S)-5α- Ergostane-3α,22-diol是首次從柳杉中分離得到。

由抗菌試驗結果得知，心材精油HO4分離部之所以具較佳的抗腐朽菌和抗植物病原菌活性，與β-Eudesmol、β-Elemol、δ-Cadinol和Ferruginol 四種化合物有關。由抗台灣家白蟻試驗結果顯示，柳杉葉子精油和甲醇抽出物均具有很強的抗白蟻活性，而葉子精油之作用機制係由揮發性和接觸毒二者共同作用而造成。比較各化合物之抗白蟻活性，證實β-Eudesmol、p-Cymene、Terpinene-4-ol、α-Terpineol、α-Pinene和γ-Terpinene 四種化合物對台灣家白蟻均有很好的毒殺效果。

由抗病媒蚊幼蟲試驗結果得知，柳杉葉子精油之LO2分離部和邊材甲醇抽出物之正已烷可溶部對埃及斑蚊幼蟲和白線斑蚊幼蟲均有很好的抑制效果。比較抽出成分之抗病媒蚊幼蟲活性，以邊材正己烷可溶部SH3分離部得到2-Methylanthraquinone和葉子精油中LO2分離部所得到Limonene、γ-Terpinene、Terpinolene、β-Myrcene、3-Carene、p-Cymene 六種化合物對埃及斑蚊和白線斑蚊幼蟲均具有很好的抑制效果，其中，2-Methylanthraquinone對二種病媒蚊幼蟲之毒殺效果最強，其效果幾可媲美商業用的陶斯松。進一步比較2-Methylanthraquinone衍生物之抗病媒蚊幼蟲活性，仍然以C-2位置上接甲基（-CH3）之2-Methylanthraquinone毒殺效果最佳。而由海蝦致死試驗結果得知，柳杉葉子精油和Limonene、γ-Terpinene、Terpinolene、β-Myrcene、3-Carene及p-Cymene 六種化合物對海蝦都有毒殺效果，顯示葉子精油和6種化合物若要作為天然殺孑孓藥劑，並不適合應用於有其他生物的水中。至於柳杉邊材甲醇抽出物及2-Methylanthraquinone對海蝦之LC50值分別大於100 μg/mL和大於50 μg/mL，顯示二者對海蝦並沒有毒性，適合開發成理想的天然殺孑孓藥劑。

關鍵字

柳杉 ； 精油 ； 抽出成分 ； 抗木材腐朽菌活性 ； 抗植物病原菌活性 ； 抗白蟻活性 ； 抗病媒蚊幼蟲活性 ； 海蝦致死試驗

並列摘要

The Japanese cedar, Cryptomeria japonica D. Don, is well-known in Taiwan as one of the important plantation tree species, thereby the potential utilizations of C. japonica are imperative study for forest management and wood production. Thus, in this study the essential oils and extracts from different parts of C. japonica were extracted with hot water and methanol, respectively, and followed by evaluating their bioactivities using various kinds of assays, including antifungal, antipathogenic, antitermitic, and mosquito larvicidal bioactivities. The pure compounds of essential oils and methanolic extracts of C. japonica were finally isolated by chromatographic separation techniques and identified by modern spectroscopic analyses, such as MS, FTIR, NMR, etc. Identified compounds were also examined with bioactive assays to confirm their bioactivities. In addition, the structure-bioactivity relationships of extractives were also elucidated.

From the results of GC and GC-MS analyses, the heartwood essential oil was found to contain mainly γ-cadinene (14.46%), epi-bicyclosesqui- phellandrene (13.15%) and δ-cadinene (12.86%); the main constituents of sapwood essential oil were isopimarol (23.11%) and ferruginol (14.74%); the bark essential oil has ferruginol (16.81%) and δ-cadinene (6.76%) as main compounds and ent-kaur-16-ene (24.90%) and β-elemol (18.84%) were the dominant components of leaf essential oil. In addition, twenty-four compounds were isolated and identified from the heartwood, sapwood, bark and leaf of C. japonica, including bisabola-7(14),10-dien-1α,4α-diol, khusinodiol, δ-cadinol, α-cadinol, γ-eudesmol, β-eudesmol, β-elemol, cryptomeridol, ferruginol, cryptojaponol, isopimaric acid, 7β-hydroxydeoxocryptojaponol, isopimarol, isopimarinol, phyllocladan-16α-ol, sandaracopimaric acid, 12-hydroxy-6,7- secoabieta-8,11,13-trien-6,7-dial, ent-kaur-16-ene, sugiol, cryptoresinol, 2-methylanthraquinone, 6β,22-dihydroxystigmasterol, (22S)-5α-ergostane- 3α,22-diol and β-sitosterol. Among them, khusinodiol, 2-methylanthraquinone, 6β,22-dihydroxystigmasterol and (22S)-5α-ergostane-3α,22-diol were first isolated from C. japonica.
From the results of antifungal assay, the HO4 fraction of heartwood essential oil had the strongest antifungal activity against four wood decay fungi and six plant pathogenic fungi. It correlates with the presence of β-eudesmol, β-elemol, δ-cadinol and ferruginol. On the basis of the results of antitermitic activity against Coptotermes formosanus Shiraki, the essential oil and methanolic extract from C. japonica leaf had the strongest termiticidal property. The termiticidal mechanisms of leaf essential oil are attributable to its volatility and toxicity. Comparison of antitermitic activity of eleven compounds revealed that β-eudesmol, p-cymene, terpinene-4-ol, α-terpineol, α-pinene and γ-terpinene showed high antitermitic activities.

Results obtained from the mosquito larvicidal tests, the LO2 fraction of leaf essential oil and Hex. sol. fraction of sapwood extract were found to be the most effective against both Aedes aegypti and Aedes albopictus larvae. Comparison of mosquito larvicidal activity of thirteen compounds revealed that 2-methylanthraquinone isolated from SH3 fraction and limonene, γ-terpinene, terpinolene, β-myrcene, 3-carene and p-cymene isolated from LO2 fraction had the best inhibitory effect against A. aegypti and A. albopictus larvae. Among these pure constituents, 2-methylanthraquinone exhibited the best mosquito larvicidal action against A. aegypti and A. albopictus larvae. The mosquito larvicidal activity of 2-methylanthraquinone was similar to that of chlorpyrifos, which is a well-known commercial insecticide. In addition, comparisons of mosquito larvicidal activity of 2-methylanthraquinone congeners demonstrated that anthraquinone skeletaton with a methyl group at C-2, such as 2-methylanthraquinone, exhibited the strongest mosquito larvicidal activity.

Based on the results of the brine shrimp toxicity assay, leaf essential oil, limonene, γ-terpinene, terpinolene, β-myrcene, 3-carene and p-cymene were demonstrated to have a high toxicity against the brine shrimp, indicating they may be toxic to other invertebrates in the river. In addition, sapwood methanolic extract and 2-methylanthraquinone tested showed a low toxicity against the brine shrimp, with a LC50 >100 μg/mL and >50 μg/mL, respectively, indicating they have good potential as a source for natural larvicides.

並列關鍵字

Cryptomeria japonica ； essential oil ； extractives ； antifungal activity ； antipathogenic activity ； mosquito larvicidal activity ； brine shrimp toxicity assay

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