Study on Antibacterial and Antioxidant Constituents of Cryptomeria japonica Bark

本研究以柳杉（Cryptomeria japonica D. Don）樹皮的乙醇抽出物進行抗細菌和抗氧化的活性評估。柳杉樹皮乙醇抽出物和其各可溶部中，以正己烷可溶部之抗細菌活性最佳，最小抑制濃度值（Minimum inhibition concentration, MIC）為250 μg/mL，因此取此可溶部進一步地分離出有效成分，並於活性的次分離部中純化鑑定出9種化合物，包括7種雙類（Ferruginol、Isopimaric acid、Iguestol、Isopimarol、Phyllocladan-16α-ol、Sandaracopimarol和Sugiol）與2種固醇類（β-Sitosterol和β-Sitostenone），β-Sitostenone為首次自柳杉中分離出的化合物。其中6種化合物具有抗細菌活性，活性依序為：Ferruginol > Isopimaric acid > Sugiol > Sandaracopimarinol > Iguestol > Isopimarol。以Ferruginol具有最佳的抗細菌活性，對所有的試驗菌種之MIC範圍為6.3-12.5 μg/mL，其次為Isopimaric acid對糞腸球菌（Enterococcus faecalis）與金黃色葡萄球菌（Staphylococcus aureus）的MIC值為15.6 μg/mL，皆具有良好的抗細菌活性。抗氧化活性評估方面，試驗結果顯示，柳杉樹皮抽出物的乙酸乙酯可溶部之總酚類含量（789.7 mg of GAE/g）、總黃酮類含量（35.8 mg of RE/g）與原花青素含量（464.1 mg of CAE/g）皆最高。此外，乙酸乙酯可溶部具有最好的DPPH捕捉能力（IC50 = 4.4 µg/mL）、總抗氧化能力（2.87 mM of Trolox）及還原能力，而抑制β-胡蘿蔔素脫色的能力亦最佳；續以層析方法對乙酸乙酯可溶部進行分離，從活性次分離部中分離出(+)-Catechin和(–)-Epicatechin，此兩種化合物均具有良好的抗氧化活性。綜合上述結果顯示，柳杉樹皮的乙醇抽出物為良好的天然抗細菌與抗氧化物質來源，值得進一步地開發與利用，以提升柳杉樹皮的利用價值。In this study, the ethanolic extract from the bark of Cryptomeria japonica D. Don was evaluated its antibacterial and antioxidant activities. Among ethanolic extract and its four fractions, n-hexane soluble fraction possessed the best antibacterial activity against four Gram-positive bacteria (Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, and Methicillin-resistant Staphylococcus aureus, MRSA) with minimum inhibitory concentration (MIC) of 250 μg/mL. Nine compounds including seven diterpenoids (ferruginol, isopimaric acid, iguestol, isopimarol, phyllocladan-16α-ol, sandaracopimarol and sugiol) and two steroids (β-sitosterol and β-sitostenone) were isolated, and it is the first time that β-sitostenone was isolated from the C. japonica plant. Six compounds exhibited excellent antibacterial activities, their ability in decreasing order was as follows: ferruginol > isopimaric acid > sugiol > sandaracopimarinol > iguestol > isopimarol. Ferruginol possessed the strongest antibacterial activity within all the compounds and had MIC values ranging from 6.3 to 12.5 μg/mL against all bacteria tested.n addition, antioxidant activities of sugi bark extract and four fractions were also examined. According to the results, the fraction with the highest content of total phenolic contents (789.7 mg of GAE/g), total flavonoid contents (35.8 mg of RE/g) and proanthocyanidin contents (464.1 mg of CAE/g) was ethyl-acetate soluble fraction. Also, ethyl-acetate soluble fraction exhibited the best DPPH radical scavenging activity (IC50 = 4.4 µg/mL), TEAC value (2.87 mM of trolox), reducing power, and the ability to decrease color bleaching of β-carotene. (+)-Catechin and (–)-epicatechin were isolated and exhibited excellent antioxidant activities. Results showed that ethanolic extract of C. japonica bark is good at antibacterial and antioxidant activities and is worthy of further explorative utilization.目 錄 I目次 III目次 V要 XIbstract XII、前言 1、文獻回顧 3、林木抽出物之抗細菌活性 3一）抽出物之抗細菌活性 4二）抽出成分之抗細菌活性 4、林木抽出物之抗氧化活性 11一）樹皮抽出物之抗氧化活性 12二）原花青素在抗氧化所扮演的角色 17、柳杉抽出成分之生物活性 20一）抗真菌活性 20二）抗細菌活性 21三）抗病媒蚊活性 22四）抗蠻活性 22(五) 抗白蟻活性22(六) 防治衣魚 23(七) 細胞毒殺試驗 23 (八) 降血脂活性 24(九) 保肝活性 24(十) 其他活性 24、材料與方法 26、試驗材料 26一）柳杉樹皮 26二）試驗藥品與溶劑 26三）試驗菌種 27、試驗方法 27一）柳杉樹皮乙醇抽出物之製備 27二）柳杉樹皮乙醇抽出物之分離 27三）化合物結構鑑定 28四）抗細菌活性試驗 28五）抗氧化活性試驗 29六）統計分析 32、結果與討論 33、柳杉樹皮抽出物之抗細菌活性 33一）乙醇抽出物及各可溶部之抗細菌活性 33二）正己烷可溶部及其各次分離部之抗細菌活性 34三）活性次分離部之化合物分離與鑑定 36四）化合物之抗細菌活性 72、柳杉樹皮抽出物之抗氧化活性 74一）乙醇抽出物及各可溶部之抗氧化活性 74二）乙酸乙酯各可溶部次分離部之抗氧化活性 85三）活性次分離部之化合物分離與鑑定 87四）化合物之抗氧化活性比較 94、結論 95、參考文獻 9