檸檬桉葉部精油及甲醇抽出物對小花蔓澤蘭種子發芽與生長抑制之研究

Abstract

小花蔓澤蘭(Mikania micrantha H.B.K)的蔓延及其對農業及生態所造成的危害，已成為全民防除的目標，本研究利用檸檬桉（Eucalyptus citriodora）的葉部，製備精油、甲醇抽出物及熱水抽出物，進行生物抑制活性評估，並進一步的分劃、分離、分析與純化，目的在探討檸檬桉是否可抑制菊科的小花蔓澤蘭(Mikania micrantha H.B.K)的繁衍及散佈，以解決過度蔓延，減少其對森林植物及環境之傷害，並做為開發天然除草劑之基本依據。 綜合試驗結果顯示，在精油抑制方面，檸檬桉精油成分對於抑制小花蔓澤蘭是具有高發芽及生長抑制活性，在培養皿抑制試驗，發現檸檬桉精油對種子發芽、胚軸及胚根生長抑制效果最佳， IC50值均<20 ppm，其次為赤桉(IC50值分別為43 ppm、402 ppm及40 ppm)，而柳杉精油則呈較低的抑制效果(IC50值分別為419 ppm、240 ppm及725 ppm)。在精油主成分對種子發芽及生長抑制方面，以含氧萜類化合物1,8-Cineole、Citronellal、Citronellol具有明顯抑制效果，其中以1,8-Cineole抑制效果最佳，發芽抑制的IC 50值<10 ppm，生長抑制的IC 50值均<50 ppm，而萜類碳氫化合物p-Cymene、α-Pinene、β-Pinene 與（+）-Aromadendrene則均呈現低的抑制效果。此外，同為含氧萜類化合物的標準品（-）-Carveol及Citral與酚類化合物Eugenol同樣具有明顯抑制效果，其中以（-）-Carveol及 Eugenol抑制效果最佳，發芽抑制率IC 50<10 ppm，生長抑制的IC 50值均<50 ppm。各精油或是精油主成分於發芽及生長抑制，在沙土種植與在培養皿有相同之趨勢，但在沙土種植時之抑制效果較差。 檸檬桉葉部甲醇抽出物抑制方面，經分劃、分離及生物抑制活性評估，發現正己烷可溶部對於發芽較具抑制活性，乙酸乙脂可溶部對於胚軸及胚根生長抑制較具活性。以HPLC分離，由H-6-7中分離鑑定出5-Hydroxy-7,4′-dimethoxy-6-methyl-flavone及p-Menthane-3,8-diol。其中，p-Menthane-3,8-diol為其主要成分，對於種子發芽及生長抑制顯著，其抑制發芽、胚軸及胚根生長IC50值分別為138 ppm、172 ppm及170 ppm，於200 ppm時對發芽、胚軸及胚根生長抑制分別可達60％、75％及60％以上，但p-Menthane-3,8-diol也是含氧萜類化合物，只是在精油中含量極少。乙酸乙脂可溶部次分離部抑制幼苗生長活性集中於EA-7次分離部，其對胚軸及胚根生長IC50值分別為1200 ppm 及 2400 ppm，抑制效果並不是非常明顯，但仍以HPLC進行分離，由EA-7中分離鑑定出5-Hydroxy-7,4''-dimethoxy-6,8-dimethylflavone。 在檸檬桉葉粉實際應用在小花蔓澤蘭之發芽及抑制試驗上，顯示檸檬桉葉粉對於小花蔓澤蘭種子或幼苗生長是具有抑制效果的。抑制種子發芽方面，前 3 週發芽抑制隨著時間的增加抑制率隨之下降，於第三週時達到最低，但可發現在第 3週之後存活的株數則開始有減少的趨勢。對於幼苗生長抑制，在施灑量大於 20 g檸檬桉葉粉/200 g培養土時，會造成幼苗完全死亡，而施灑量 10 g 及 5 g 的組別，雖未完全死亡，但其存活株數已有明顯差異，施灑量5 g 的組別，莖及根的相對抑制率分別為52％及43％。 綜合以上試驗結果指出，檸檬桉之葉部精油及萃取物是含有相剋作用化合物的，可抑制小花蔓澤蘭種子發芽及幼苗生長，且檸檬桉葉部精油及其精油成分對小花蔓澤蘭種子發芽及幼苗生長抑制有明顯的效果，與精油相比，甲醇抽出物則需要較高的濃度才可達到抑制效果，但若將精油及甲醇抽出物共同使用，能有某種程度上的加成作用。

【關鍵詞】小花蔓澤蘭、生物性雜草控制、檸檬桉、相剋作用、相剋化學藥劑、精油

Abstract Because the widespread mile-a-minute (Mikania micrantha H.B.K) weed become a threat to agriculture and ecosystem, it has become a target of eradication for all people. In this study, we used the leaves of Eucalyptus citriodora to prepare essential oil, methanolic and hot-water extracts, and used bioactivities evaluations to further fractionate, isolate, analyze and purify the active ingredients to examine whether the species could deter the propagation and spread of the Compositae weed and reduce its damage towards forest plants and environments. The endeavor was based on the premise of developing effective herbicides from nature. The overall experimental results indicated that on the inhibitory effects of essential oils, the leaf essential oil of E. citriodora had high germination and growth inhibition efficacies against M. micrantha. In Petri-dish tests, the essential oil was particularly effective in inhibiting the germination, growth of hypocotyle, and growth of radicle, with IC50 values all < 20 ppm. The leaf oils of E. camaldulensis, on the other hand, had IC50 values of 43, 402, and 40 ppm, respectively; Leaf oil of Cunninghamia lanceolata, with lower inhibitory efficacies had IC50 values of 419, 240, and 725 ppm, respectively. .As for the main ingredients of the essential oils in inhibiting seed germination, the oxygenated terpenoides of 1,8-cineole, citronellal, citronellol all had significant inhibitory efficacy. In particular, 1,8-cineole was the most effective, which inhibited seed germination at IC50 < 10 ppm and suppressed both embryonic growths at IC50 < 50 ppm. The hydrocarbon terpenoids such as p-cymene, α-pinene, β-pinene, and …..(+)-aromadendrene all exhibited low inhibitory efficacies. Furthermore, oxygenated terpenoid standards such as (-)-carveoland citral, and phenolics such as eugenol, although not in the leaf oils, did show strong inhibitory effects. (-) carveol and eugenol, in particular, had germination inhibition dosage of IC 50<10 ppm and both growth inhibition dosages of IC 50<50 ppm. The inhibitory effects showed the identical trends when comparing tests on Petri-dishes and on a sandy-loam plot. However, the efficacies were weaker on the latter. The fractionated, isolated methanolic extracts of E. citriodora leaves were also tested for their inhibitory bioactivities. The n-hexane extract was found to be more germination inhibitory against M. micrantha, whereas the ethyl acetate (EA) extract was active in suppressing the growth of hypocotyle and radicle. Upon isolation by HPLC, 5-hydroxy-7,4’-dimethoxy-6-methyl-flavone and p-menthane-3,8-diol were found in the H-6-7 peak. The p-menthane-3,8-diol was the main ingredient and showed significant inhibitory efficiencies against both seed germination and hypocotyle and radicle growths, with IC50 values of 138, 172 and 170 ppm, respectively. At a dosage of 200 ppm, the suppression rates were 60%, 75% and 60%, respectively. Notably, p-menthane-3,8-diol is an oxygenated terpene, which rarely appear in essential oils. The active EA subfraction was concentrated in the EA-7 peak, which had IC50 values against the growths of M. micrantha hypocotyle and radicle .were 1200 and 2400 ppm, respectively. Although the inhibitory effect was not distinct, the HPLC isolated from EA-7 5-hydroxy-7,4''-dimethoxy-6,8-dimethyl flavone. In a field test applying E. citriodora leaf powder on the seed germination and growth of M. micrantha, inhibitory effect was observed. With regard to seed germination, the efficacies decreased with time in the first 3 wks and reached minimum at the 3rd wk. However, the surviving seedling number had a tendency to decrease after the 3rd wk. As for the growth suppression effect, at dosages greater than 20 g of leaf powder to 200 g of cultivating soil, all seedlings died. Whereas for dosages of 10 and 5 g, although total annihilation was not achieved, but the surviving number already showed significant differences. The 5 g group had root and stem growth depressed to 52% and 43%, respectively. Summarizing the above results, the leaf essential oil and extracts of E. citriodora contained fair amounts of allelopathic substances to inhibit the seed germination and seedling growth of M. micrantha. Furthermore, its leaf oil and oil ingredients hade marked inhibitory effects unlike those of the leaf methanolic extracts which were inhibitory only at much higher concentrations. If both were applied simultaneously, a synergistic effect was observed.

【Key words】Mikania micrantha, biological prevention, Eucalyptus citriodora, allelopathy, allelochemicals, essential oil