四種桉樹葉精油組成及生物活性之探討

Bioactivity of Essential Oils from Leaves of Four Eucalyptus species

何振隆(Chen-Lung Ho)

指導教授 ： 張上鎮

共同指導教授 ： 蘇裕昌(Yu- Chang Su)

國立臺灣大學/生物資源暨農學院/森林學研究所/碩士(2004年)

https://doi.org/10.6342/NTU.2004.01114

摘要

桉樹為世界主要之紙漿用材，分布廣泛，引進台灣已有百年歷史。為提高桉樹多方面應用，本研究以四種不同種類之桉樹葉片，即垂尾桉 (Eucalyptus urophylla) 、玫瑰桉 (E. grandis) 、赤桉 (E. camaldulensis) 及檸檬桉 (E. citriodora) 等，以傳統水蒸氣蒸餾法萃取桉樹葉精油，並分離精油組成成分及進一步評估其生物活性，如：抗氧化、抗菌 (細菌、黴菌及木材腐朽菌)等活性。
精油收率依序為赤桉 (3.48 ml/100g)、垂尾桉 (3.14 ml/100g)、玫瑰桉 (3.01 ml/100g)及檸檬桉 (1.89 ml/100g)。各精油的成分化合物鑑定，垂尾桉葉精油共鑑定54個化合物，最主要主成分為γ-Terpinene。玫瑰桉葉精油共鑑定67個化合物，最主要主成分為1,8-Cineole。赤桉葉精油共鑑定55個化合物，主要主成分為1,8-Cineole。檸檬桉葉精油共鑑定40個化合物，最主要成分為Citronellal。
抗氧化試驗結果顯示，應用硫氰酸鐵法於72 hr，測定四種桉樹葉精油(200 μg/ml)抑制亞麻油酸氧化能力為55.81-82.57%。DPPH自由基捕捉能力之IC50，垂尾桉398 μg/ml，玫瑰桉544 μg/ml，檸檬桉559 μg/ml，赤桉718 μg/ml。 Rapid screening of antioxidant by dot-blot and DPPH staining測試法中所測定之b*值，與DPPH自由基捕捉能力之值進行迴歸比較，R2值可達0.93，顯示二者有極高相關性，故證實Rapid screening of antioxidant by dot-blot and DPPH staining法可應用於DPPH捕捉自由基能力之快速篩選測試方法。除此之外，我們再將四種桉樹葉精油以TLC片法分離之，並浸漬於DPPH溶液後測試各分離部之b*值，且以GC-MS鑑定所含化合物，確認四種桉樹主要捕捉自由基之化合物，在垂尾桉葉精油中以γ-Terpinene、Thymol等化合物最強，玫瑰桉精油中，以Thymol化合物為最強，赤桉葉精油中，以1,8-Cineole、Terpinene-4-ol等化合物，檸檬桉葉精油中，以Citronellol、Terpineolene等化合物，這些具捕捉自由基能力化合物，均以上述Dot-blot and DPPH staining測試法加以證實。在還原能力方面，垂尾桉 (還原效果相當於22.33 μg/ml的BHT) 、玫瑰桉 (19.85 μg/ml) 、赤桉(16.80 μg/ml) 、檸檬桉 (18.26 μg/ml) ，各葉精油對自由基捕捉能力與還原力大小成正相關。螯合亞鐵離子能力，垂尾桉葉精油對螯合亞鐵離子能力為66.4%、玫瑰桉60.8%、赤桉33.2%、檸檬桉22.8%。總酚類化合物含量分析結果得知，垂尾桉總酚量為24.21 mg GAE/g essential oil，玫瑰桉為22.80、檸檬桉為16.92、赤桉為13.60。再將總酚類化合物含量與硫氰酸鐵法、DPPH自由基捕捉能力及還原能力予以迴歸，發現R2高達0.90以上，但與螯合亞鐵離子能力相關性最差，R2僅為0.79，但趨勢仍然一致。
抗細菌試驗則採用了紙錠擴散法及肉湯稀釋法等二法進行試驗，菌種為使用革蘭氏陰性菌的大腸桿菌 (Escherichia coli) 、肺炎桿菌 (Klbesiella pneumoniae)、綠膿桿菌 (Pseudmonas aeruginosa)，以及革蘭氏陽性菌的金黃色葡萄球菌 (Staphylococcus aureus) 等四種菌株。無論對陽性菌與陰性菌均以赤桉抗細菌活性最強，其次為垂尾桉，再其次為檸檬桉，最弱為玫瑰桉精油。而其抗細菌活性大小與主成分1,8-Cineole含量多寡並無相關性，且對於其他成分，如：γ-Terpinene、p-Cymene、Citronellal、Isopulegol、Citronellol等亦無明顯相關，至於精油微量成分方面，則無相關關係，故推論可能其抗細菌活性為來自於二種或多種精油組成分結合，方可發揮抗菌幼纂C
　　抗黴菌試驗結果顯示四種桉樹葉精油中，以檸檬桉抗黴菌效果為最佳，且對各試驗黴菌廣泛有效，而以垂尾桉抗黴菌效果最差。抗腐朽菌試驗結果亦顯示檸檬桉精油之抗腐朽菌活性最高，可做為極佳之抗木材腐朽劑。主要原因為檸檬桉葉精油主成分Citronellol與Citronellal等二化合物，此二化合物的存在對於抗黴菌及抗腐朽菌活性有明顯的助益。

關鍵字

抗細菌活性 ； 抗真菌活性 ； 精油 ； 抗氧化活性

並列摘要

Eucalyptus is an important pulpwood genus globally which has wide distribution and was introduced to Taiwan over a century ago. In order to diversify utilization of the eucalypts, essential oils from the leaves of 4 species, i.e., E. urophylla, E. grandis, E. camaldulensis, and E. citriodora were extracted using traditional steam distillation. The components of the essential oils were analyzed and isolated. Furthermore, their biological activities, such as antioxidative, antimicrobial (including bacteria, mildews and wood decaying fungi) functions were examined.
Yields of essential oils were in the order: E. camaldulensis (3.48 ml/100g), E. urophylla (3.14 ml/100g), E. grandis (3.01 ml/100g) and E. citriodora (1.89 ml/100g). Components identification of the individual essential oils entailed that E. urophylla had 54 identified compounds, with γ-terpinene predominant; E. grandis had 67 identified compounds, and the richest Fraction was 1,8-cineole; 55 compounds were identified from the essential oil of E. camaldulensis, which had 1,8-cineole as the main component; and from the E. citriodora essential oil, 40 compounds were identified, and citrionellal was the dominant component.
The antioxidant tests indicated that by using the ferric thiocyanate method for a test period of 72 h, capabilities of 200 μg/ml of the 4 essential oils to suppress oxidation of linoleic acid were between 55.81% and 82.57%. The IC50 values of DPPH free radical scavenging capabilities were in the order E. urophylla, 398 μg/ml; E. grandis, 544 μg/ml; E. citriodora 559 μg/ml; and E. camaldulensis 718 μg/ml. Regressions between the b* values determined in the rapid screening of antioxidant by dot-blot and DPPH staining technique and the DPPH free radical scavenging capability values gave a R2 of 0.93, indicating a very significant correlation between the 2 methods. In addition, we separated components of the 4 essential oils by TLC plate method. The main free radical scavenging compounds in the 4 eucalypts were found to be γ-terpinene and thymol in E. urophylla; thymol in E. grandis; 1,8-cineole, and terpinene-4-ol in E. camaldulensis; and citronellol and terpineolene in E. citriodora as the most active ingredient. Analysis of the total phenolic content and the antioxidative capabilities gave 24.21, 22.80, 16.92, and 13.60 mg GAE/g essential oils for E. urophylla, E. grandis, E. citriodora, and E. camaldulensis, respectively. When the results of total phenolic content were compared to those of ferric thiocyanate, DPPH free radical scavenging capacities, and reductive capacities, R2 values of 0.90 or higher were obtained.
Paper disk diffusion and broth dilution methods were adopted for the assay of antibacterial activities. Regardless of the bacterial types, E. camaldulensis essential oil showed the strongest antibacterial activity, which was followed by the essential oils of E. urophylla, and E. citriodora; and that of the E. grandis was the least effective. The antibacterial activities were found to have no correlation with the amounts of main component, 1,8-cineole, neither was there any significant correlation with the other compounds, such as γ-terpinene, p-cymene, citronellal, isopulegol, citronellol etc. Thus, we stipulate that the antibacterial activity may have its efficacy arisen largely from the combined effects of 2 or more essential oil components.
Experimental results of the anti-mildew tests of the 4 eucalypts essential oils indicate that E. citriodora had the best efficacy, and were extensively effective against all tested mildews. E. urophylla, on the other hand, had the poorest efficacies. On the anti wood decay fungi tests, the results also show that E. citriodora essential oil had the best antifungal activity, and should perform as an excellent wood preservative. The main reason for its effectiveness was due to the presence of citronellol and citronellal compounds. The 2 compounds had obvious benefit in fighting fungi.

並列關鍵字

essential oil ； antibacterial ； antifun ； antioxidant

參考文獻

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