臺灣杉心材抗真菌成分之分析、鑑定及其抑菌機制

Analyses and Identification of Antifungal Compounds Isolated from the Heartwood of Taiwania cryptomerioides and Their Mechanisms

吳季玲(Chi-Lin Wu)

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(2005年)

https://doi.org/10.6342/NTU.2005.02789

摘要

臺灣杉為臺灣原產重要經濟樹種，亦是重要的造林樹種之一。就木材性質而言，具有極佳的耐腐朽性及抗蟻性，與著名的檜木類木材相當，同屬性能優越的材種。本研究以甲醇溶劑萃取出臺灣杉心材的抽出成分，配合抗菌活性試驗之篩選，分離、純化及鑑定出具抗真菌活性之主要成分，亦將具活性之化合物的化學結構予以修飾，期能由相同骨架之化合物中，合成出具較佳生物活性之化合物。除此之外，並利用蛋白質體分析方法且配合質譜分析技術、DNA斷裂分析、西方轉漬法等，瞭解化合物抑制真菌細胞生長作用之機制。

本研究由臺灣杉心材中，經由抗真菌活性試驗，篩選出正己烷可溶部及乙酸乙酯可溶部，共分離出12個化合物：α-Cadinol, T-Cadinol, T-Muurolol, α-Cedrol, Hinokiol, Sugiol, Ferruginol, Isopimarinol, Taiwanin A, Helioxanthin, Savinin及Taiwanin C。此外，本研究將3種Cadinane類倍半萜進行結構之修飾，共分離純化出13個Cadinane類倍半萜化合物： 15-Oxo-T-cadinol 、3-Oxo-T-cadinol 、3-Oxo-15-hydroxy-T-cadinol、15-Oxo-T-muurolol、3-Oxo-T-muurolol、3β-Hydroxy-T-muurolol、3β-Ethoxy-T-muurolol、15-Oxo-α-cadinol、3-Oxo-α-cadinol、3-Oxo-15-hydroxy-α-cadinol、4ξH-Cadinan-10α-ol、4ξH-Cadinan-10β-ol、4ξH-Muurolan-10β-ol。

由抗真菌活性試驗結果證實，臺灣杉心材正己烷次分離部之所以具較佳之抗腐朽菌活性，是與α-Cadinol、T-Cadinol及T-Muurolol等Cadinane類化合物有關，尤其是具有極佳抗腐朽菌活性之α-Cadinol。此外，乙酸乙酯次分離部之抗腐朽菌活性，推測是與Ferruginol、Savinin及Taiwanin C有關。此外，臺灣杉心材抽出成分中，α-Cadinol與Ferruginol之抗植物病原真菌活性最佳。

Cadinane倍半萜類化合物的結構與抗菌活性關係(Quantitative
structure-activity relationship, QSAR)之結果顯示，Cadinane骨架中兩個六碳環相接位置上的氫為trans構形與C-10位置上之赤道向羥基具最強的抗腐朽活性，而且C-10位置上之羥基對於抗腐朽菌活性之影響小於trans構形。此外，Cadinane骨架的化合物中含雙鍵及C-3與C-4位置之官能基和抗腐朽菌活性有相關性，且化合物之疏水性亦為抗菌活性的重要影響因子之一。

蛋白質體分析試驗結果顯示，α-Cadinol所影響Trametes versicolor菌絲體生長的蛋白質表現中，包括有與真菌受傷害有關之熱休克蛋白質(Heat shock protein, HSP70) ， Virulence-regulating 蛋白質和DNA-damage inducible蛋白質，及與程式性細胞死亡(Programmed cell death, PCD)有關之Caspase-3、Protease和Cytochrome c，其表現量皆增加。經由DNA斷裂分析、以及與程式性細胞死亡相關蛋白質作免疫轉漬反應等結果證實，α-Cadinol會引發T. versicolor程式性細胞死亡，而達到抗菌之功效。

關鍵字

臺灣杉 ； 心材 ； 耐腐朽性 ； 抗真菌成分 ； 結構鑑定 ； 光譜分析 ； Cadinane倍半萜類 ； 蛋白質體分析 ； 抑菌機制

並列摘要

Taiwania (Taiwania cryptomerioides Hayata), named after its native island of Taiwan, is one of the most economically important tree species grown in Taiwan. As for decay resistance, Taiwania is classified as a species with an excellent durability in Taiwan.

Twelve compounds were isolated using bioassay-guided fractionation of the methanolic extract from the heartwood of T. cryptomerioides. Antifungal properties of 12 compounds against wood-rot fungi were estimated in vitro. According to Mass, 1H-NMR, 13C-NMR, and 2D-NMR analysis, their structures were identified, namely α-cadinol, T-cadinol, T-muurolol, α-cedrol, hinokiol, sugiol, ferruginol, isopimarinol, taiwanin A, helioxanthin, savinin and taiwanin C. Moreover, 13 cadinane sesquiterpenes compounds, namely 15-oxo-α-cadinol, 3-oxo-α-cadinol, 3-oxo-15-hydroxy-α-cadinol, 15-oxo-T-cadinol,
3-oxo-T-cadinol, 3-oxo-15-hydroxy-T-cadinol, 3β-ethoxy-T-muurolol, 15-oxo-T-muurolol, 3-oxo-T-muurolol, 3β-hydroxy-T-muurolol, 4ξH-cadinan-10α-ol, 4ξH-cadinan-10β-ol、4ξH-muurolan-10β-ol were isolated and identified.

Quantitative structure-activity relationships of these candinanes have been investigated. Further comparison of the molecular configuration of these cadinanes reveals that cadinane skeletal sesquiterpenoids with an equatorial hydroxyl group at C-10 and a trans configuration at the ring junction, such as the case for α-cadinol, exhibited the strongest antifungal activity. The correlation was observed between the antifungal activity of the tested compounds and log P. Furthermore, the presence of theunsaturated double bond and oxygen-containing functional group in the compounds plays a key role in their antifungal activity. In addition, the stereo configuration of cadinane sesquiterpenes also influences their antifungal activity.

Results of proteomic analyses showed that α-cadinol influenced the expression of proteins from Trametes versicolor mycelia. Those over expression proteins identified include heat shock protein (HSP70),
virulence-regulating protein and DNA-damage inducible protein which are related to the damage of cells. Furthermore, the over expression proteins including caspase-3, protease and cytochrome c associating with
programmed cell death (PCD) were also identified. Results obtained from the analyses of DNA fragmentation and immunoblotting with proteins related to the programmed cell death demonstrated that the antifungal performance of α-cadinol against T. versicolor is dueto the induced programmed cell death.

並列關鍵字

Taiwania cryptomerioides ； Heartwood ； Decay resistance ； Antifungal Compound ； Structure identification ； Spectra analysis ； Cadinane sesquiterpenes ； Proteomic analyses ； Antifungal mechanism

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