知母抗黑斑病菌活性成分分離與純化

Study on the Anti-Alternaria brassicola Principles from the Rhizomes of Anemarrhena asphodeloides.

黃兆宇(Chao-Yu Huang)

指導教授 ： 石麗仙

共同指導教授 ： 羅朝村

國立虎尾科技大學/電機資訊學院/光電與材料科技研究所/碩士(2010年)

摘要

植物源農藥是由植物體內萃取用於防治作物病蟲害的製劑。本實驗以黑斑病菌(Alternaria brassicola, Ab)和炭疽病菌(Colletortichum higginsianum, Ch)為測試菌株，進行中草藥抗真菌活性試驗，其中知母(Anemarrhena asphodeloides)甲醇萃取物在1000 ppm 於六天平均時分別對炭疽病菌和黑斑病菌有87%和94%的生長抑制率。
知母(A. asphodeloides)為百合科(Liliaceae)植物知母的根，產於中國、韓國和日本，為一常用傳統中藥。研究證實知母具有抗糖尿病、抑制血小板凝集、利尿、軟體動物毒殺、抗真菌和抗酵母菌等活性。
由知母根部經鑑定後粉碎，以甲醇熱迴流抽取，甲醇抽取物以水與氯仿和正丁醇進行分配萃取，其中正丁醇層在2000 ppm於六天平均時對黑斑病菌有49%抗菌活性，而其在1000 ppm於六天平均時對炭疽病菌有77%抗菌活性，因此依活性導向進行正丁醇層之抗真菌成分分離。以大孔洞樹酯、矽膠管柱層析和逆相管柱層析等方法分離出十四個化合物。經光譜解析及文獻比對各化合物光譜數據，確認八個化合物的結構，分別為mangiferin (1)、nyasol (2)、anemarrhenasaponin IV (3)、timosaponin A-III (4)、timosaponin P (5)、timosaponin Q (6)及anemarrhenasaponin I (7) 和anemarrhenasaponin Ia (8)之混合物，尚有6個化合物正進行構造解析。其中timosaponin P (5)和timosaponin Q (6)為首次分離得到之新化合物。
取純化合物進行抗菌活性實驗，其中ANAS-B-6-6及ANAS-B-11C在2000 ppm於十四天後對黑斑病菌分別有72.9%及77.5%的抑制率，其抑菌機制則有待進一步探討。

關鍵字

知母 ； 黑斑病菌 ； 炭疽病菌

並列摘要

Botanical pesticide is a formula, which was extracted from plants, use to prevent and control the plant disease. In order to examine the Chinese herbal medicine extract for their potential in inhibiting plant pathogenic fungi, Alternaria brassicola (Ab) and Colletortichum higginsianum (Ch) were selected as on indicative fungus. In our preliminary experiment, the results showed that the methanolic extract of Anemarrhena asphodeloides exhibited 87% and 94% inhibition to Ch and Ab at 1000 ppm (6-day).
A. asphodeloides, belongs to the family Liliaceae, was distributed in China, Korea and Japan. The roots of A. asphodeloides was also a Chinese tradition medicine. It was used for anti-diabetic, anti-platelet aggregation, diuretic, molluscicidal, anti-fungal and anti-yeast activities.
The roots of A. asphodeloides were powdered and extracted with methanol. The methanolic extract was concentrated under reduced pressure, and obtained brown syrup. The syrup was dissolved in water, then partitionated with CHCl3 and n-butanol to obtain CHCl3-, n-butanol-, and water-soluble layers, separatedly. According to the bioassay results, the n-butanol-soluble layer exhibited 49% inhibition to A. brassicicola at 2000 ppm (6-day) and 77% inhibition on Ch at 1000 ppm (6-day). Therefore, the n-butanol-soluble layer was selected in this investigation.
Fractionation and chromatography of the n-butanol-soluble layer of the roots of A. asphodeloides afforded fourteen compounds. Eight of them was named: mangiferin (1)、nyasol (2)、anemarrhenasaponin IV (3)、timosaponin A-III (4)、timosaponin P (5)、timosaponin Q (6)、mixture of anemarrhenasaponin I (7) and anemarrhenasaponin Ia (8). Their structure were determined by spectroscopic analysis and compared with the literature data. Timosaponin P (5) and Q (6) were new compounds.
The isolated compounds were subjected to evaluated the anti fungi activity. The result showed ANAS-B-6-6, and 11C exhibited 72.9% and 77.5% inhibition on Ab at 2000 ppm (14-day).

並列關鍵字

Alternaria brassicola ； Colletotrichum higginsianum ； Anemarrhena asphodeloides

參考文獻

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