Study on Antifungal Compounds of Michelia formosana

烏心石（Michelia formosana (Kanehira) Masamune & Suzuki）為台灣原生樹種，屬於台灣闊葉五木之一。本研究目的為評估抽出成分對於烏心石木材耐腐朽性的影響，分析心材抽出物及葉子精油中的抗腐朽菌活性化合物。試驗結果證實心材的抗腐朽性極佳，心材經乙醇萃取後顯示試材的重量損失率明顯上升，對於腐朽菌L. betulina與L. sulphureus的抗腐朽性明顯降低，表示乙醇抽出成分對心材的抗腐朽性具有重要的影響。評估烏心石各部位乙醇抽出物抑制腐朽菌的活性，其活性依序為心材＞葉部＞邊材＞樹皮，證實心材的乙醇抽出物具有良好的抗腐朽菌活性。 進一步評估心材抽出物中各可溶部的活性，以正己烷可溶部的抗菌活性最佳，其次是正丁醇可溶部、乙酸乙酯可溶部及水可溶部，顯示具抑制腐朽菌活性的心材抽出成分主要存在於正己烷可溶部。進一步由活性次分離部中分離鑑定一種倍半萜類化合物及二種生物鹼，分別是(+)-epi-Cubenol 、Annonbraine及Liriodenine。其中(+)-epi-Cubenol 與Annonbraine為首次由烏心石中分離所得之天然化合物。分析Liriodenine的抗腐朽菌活性，發現Liriodenine對L. betulina與T. versicolor等白腐菌的IC50分別為0.76與3.50 μg/mL；而對L. sulphureus、G. trabeum與F. pinicola褐腐菌的IC50均低於2 μg/mL，顯示Liriodenine具有極佳的抗腐朽菌活性。 烏心石葉精油亦具有抗腐朽菌活性，由活性分離部獲得五種化合物分別是4, 5-Epoxy-β-caryophyllene、Linalool、Guaiol、Bulnesol及β-Elemol，其中以Guaiol、Bulnesol及β-Elemol的活性較佳，其中，Guaiol與β-Elemol對L. betulina白腐菌與L. sulphureus褐腐菌的半數抑制濃度均低於50 μg/mL。 綜合上述試驗結果，本試驗由烏心石心材萃取物及葉子精油中共獲得4種具抗腐朽菌活性的成分―Liriodenine、Guaiol、Bulnesol及β-Elemol，可以有效抑制白腐菌與褐腐菌的生長，值得進一步的開發與利用。Michelia formosana (Kanehira) Masamune & Suzuki is one of the five precious hardwood trees in Taiwan. The objectives of this study were to evaluate the influence of extractives on the decay resistance of M. formosana wood and analyze the antifungal compounds of heartwood extract and leaf oil. After decay tests with white rot fungus Lenzites betulina and brown rot fungus Laetiporus sulphureus, weight losses of heartwood specimens were less than 2% against 2 kinds of rot fungi. Heartwood demonstrated high decay resistance against wood rots. However, weight losses of extracted heartwood specimens were increased significantly after decay test. It indicated that the ethanolic extract had a strong influence on the decay resistance of heartwood. Antifungal activities of ethanolic extracts obtained from M. formosana were in the following order: heartwood > leaf > sapwood > bark. Result inferred that the heartwood ethanolic extract exhibited the highest antifungal activity. Further more, antifungal activities of n-hexane, EtOAc, n-BuOH and H2O-soluble fractions obtained from heartwood ethanolic extract were assessed, the activities observed were in the order of n-hexane > n-BuOH > EtOAc > H2O-soluble fractions. These results indicated that the antifungal compounds mainly existed in the n-hexane-soluble fraction. One sesquiterpenoid, (+)-epi-cubenol, and two alkaloids, liriodenine and annonbraine, were isolated from active subfractions. (+)-epi-Cubenol and annonbraine were isolated from M. formosana for the first time. According to the antifungal test of liriodenine, IC50 values of liriodenine against white rot fungi L. betulina and T. versicolor were 0.76 and 3.50 μg/mL, respectively. And IC50 values of liriodenine against brown rot fungi L. sulphureus, G. trabeum and F. pinicola were all lower than 2.0 μg/mL. It demonstrated that liriodenine has an excellent antifungal activity. Five compounds, 4, 5-epoxy-β-caryophyllene, linalool, guaiol, bulnesol and β-elemol, were isolated from the active fractions of leaf essential oil, which also possesses antifungal activity. Amoung these compounds, guaiol, bulnesol and β-elemol have better antifungal activities against white rot fungus L. betulina and brown rot fungus L. sulphureus. According to the above results, four antifungal compounds, liriodenine, guaiol, bulnesol and β-elemol, were obtained from heartwood extract and leaf essential oil of M. formosana. These compounds which could effectively inhibit the growth of white rot fungi and brown rot fungi, are of great potential as natural wood preservatives.目錄……………………………………………………………………… Ⅰ 表目次…………………………………………………………………… Ⅴ 圖目次…………………………………………………………………… Ⅶ 摘要……………………………………………………………………… і Abstract…………………………………………………………………… ііі 壹、緒言………………………………………………………………… 1 貳、文獻回顧…………………………………………………………… 3 一、林木之天然抗腐朽性……………………………………………… 5 二、危害木材之真菌種類……………………………………………… 8 (一)、白腐菌…………………………………………………………… 11 (二)、褐腐菌…………………………………………………………… 13 (三)、軟腐菌與木材變色菌…………………………………………… 14 三、腐朽菌降解木材的可能機制……………………………………… 15 四、抽出成分之抗腐朽菌活性………………………………………… 20 (一)、聚酚類…………………………………………………………… 20 (二)、萜類……………………………………………………………… 22 (三)、生物鹼…………………………………………………………… 24 (四)、其他化合物……………………………………………………… 26 五、烏心石抽出成分之研究…………………………………………… 26 參、材料與方法………………………………………………………… 31 一、試驗材料…………………………………………………………… 31 (一)、烏心石…………………………………………………………… 31 (二)、試驗菌種………………………………………………………… 31 (三)、培養基…………………………………………………………… 32 (四)、化學試藥………………………………………………………… 32 二、試驗方法…………………………………………………………… 33 (一)、木材防腐性能試驗……………………………………………… 33 (二)、精油萃取………………………………………………………… 34 (三)、乙醇抽出物之萃取……………………………………………… 34 (四)、液相-液相分配…………………………………………………… 34 (五)、管柱層析………………………………………………………… 35 (六)、液相層析………………………………………………………… 36 (七)、抗腐朽菌試驗…………………………………………………… 36 (八)、生物鹼試劑……………………………………………………… 37 (九)、化合物結構鑑定………………………………………………… 37 (十)、Liriodenine之定量分析…………………………………………… 38 (十一)、統計分析……………………………………………………… 38 肆、結果與討論………………………………………………………… 39 一、烏心石心材抽出成分之抗腐朽菌活性…………………………… 39 (一)、抽出成分對烏心石木材抗腐朽性之影響……………………… 39 (二)、烏心石乙醇萃取物之抗腐朽菌活性…………………………… 40 (三)、心材乙醇萃取物之抗腐朽菌活性……………………………… 42 (四)、化合物之分離與鑑定…………………………………………… 45 1. 化合物(+)-epi-Cubenol之分離與鑑定……………………………… 45 2 化合物Annonbraine之分離與鑑定.………………………………… 52 3. 化合物Liriodenine之分離與結構鑑定.…………………………… 59 (五)、化合物之抗腐朽菌活性………………………………………… 68 二、烏心石葉子精油之抗腐朽菌活性評估…………………………… 75 (一)、葉子精油之抗腐朽菌活性……………………………………… 75 (二)、葉子精油各分離部之抗腐朽菌活性…………………………… 76 (三)、活性成分分離部之成分分離與鑑定…………………………… 78 1. 化合物4, 5-Epoxy-β-Caryophyllene之分離與鑑定………………… 79 2. 化合物Linalool之分離與鑑定……………………………………… 85 3. 化合物Guaiol之分離與鑑定……………………………………… 89 4. 化合物Bulnesol之分離與鑑定…………………………………… 95 5. 化合物β-Elemol之分離與鑑定…………………………………… 101 (四)、葉子精油成分之抗腐朽菌活性………………………………… 105 伍、結論………………………………………………………………… 109 陸、引用文獻…………………………………………………………… 11