醉马草-内生真菌共生体生物碱的活性研究

Drunken horse grass (Achnatherum inebrians) is a toxic perennial bunchgrass, which is so-named because it is associated with the narcosis of grazing animals on native grasslands in the northwest of China. This species is usually infected by the fungal endophyte Neotyphodium gansuense. A series of experiments were conducted to study the biological activities of alkaloids extracted from the endophyte-infected (E+) grass. The alkaloid contents of Peramine, Loline and Lolitrem B were detected by high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). Pure samples of these alkaloids from the E+ plants were isolated by extraction with ethanol and other solvents, column chromatography on silica gel, recrystallization, and single crystal cultivation. Structural determinations for these compounds were made on the basis of IR, MS, 1D-NMR, and 2D-NMR. The insect virulence measurements were carried out by using pure alkaloids for determining their respective minimum lethal concentrations by the slide-dip method. The relationship between the alkaloid concentrations and the mortality of the insects was analyzed to obtain a regression equation, then the median lethal concentration (LD50) was calculated by that equation. Insect response to the respective alkaloid concentrations at various locations on E+ plants were determined. Alkaloid contents were detected before or after pest stresses to investigate the effect of this stimulus on the levels of alkaloid production in E+ Achnatherum inebrians. The alkaloids were extracted in two stages using a two-phase solvent system and were quantified on an Agilient 1100 HPLC. The antifungal activities of the pure alkaloids from E+ plants were tested against Alternaria alternata, Bipolaris sorokiniana, Curvularia lunata, Fusarium avenaceum, Fusarium solani and Trichoderma viride. The alkaloids inhibited fungal mycelium growth, spore germination and the lengths of germ tube were also measured. The median inhibitory alkaloid concentration (IC50) on spore germination was calculated using a linear relation between the inhibitory rates and the concentration logarithm. The cytotoxicity of the pure alkaloids on animal smooth muscle cells were measured by the MTT method and enzyme-linked immunosorbent assay. The relationship between the alkaloid concentrations and the cell inhibition rates was analyzed to obtain a regression equation. The median lethal concentration (LD50) was then calculated from that equation. Future studies will determine cellular mechanisms for the insect toxicity of E+ drunken horse grass.

醉马草(Achnatherum inebrians)为禾本科芨芨草属的多年生草本植物，是我国北方天然草原主要的烈性毒草之一，与甘肃内生真菌(Neotyphodium gansuense Li et Nan)形成互惠共生体并且产生生物碱是其对家畜致毒的原因。本研究通过色谱和质谱等方法检测醉马草-内生真菌共生体中波胺(Peramine)、黑麦草碱(Loline)和Lolitrem B等生物碱的含量，结合乙醇提取、各种溶剂萃取、硅胶柱层析、重结晶、单晶培养等有机化学方法进行单体分离。通过玻片浸渍法进行昆虫毒力测定，明确纯品生物碱对供试害虫的最小致死浓度和中毒剂量，就生物碱浓度和昆虫死亡率进行回归分析并确定半致死浓度。明确生物碱对植物病原真菌菌落生长、孢子萌发和芽管伸长的抑制作用并确定其半抑制浓度。通过MTT染色法和酶标法测定纯品生物碱对正常动物细胞生长的抑制率并明确生物碱对正常细胞的半抑制浓度。

醉马草(Achnatherum inebrians (Hance) Keng)为禾本科(Gramineae)芨芨草属的多年生草本植物，是我国北方天然草原主要的烈性毒草之一，与甘肃内生真菌(Epichloё gansuensis Li et Nan)形成互惠共生体且产生生物碱是其对家畜致毒的原因。通过高效液相色谱(HPLC)法从醉马草-内生真菌共生体中检测到了Peramine及其同分异构体Homoperamine、Lolitrem B、Ergine、Ergonovine和Ergovaline6种生物碱。分离自醉马草-内生真菌共生体上的不同菌株(SN、XH和YZ)产碱种类和含量表现出多样性。从被内生真菌E. gansunensis侵染的醉马草中提取分离获得了纯品化合物，通过一系列的谱图(高分辨质谱、氢谱、碳谱、二维核磁等)以及X射线单晶衍射分析，最终确定其结构为麦角类生物碱及其同分异构体-异麦角新碱。结果表明这两种麦角生物碱有很好的杀虫活性，尤其是对禾谷缢管蚜(Rhopalosiphum padi) 和甘蓝蚜(Brevicoryne brassicae)有良好的杀虫效果，半致死浓度分别为0.125 和0.211 mM。麦角新碱和异麦角新碱对朱砂叶螨群体具有良好的杀虫效果，其半致死浓度分别为0.087 mM和0.069 mM。在大田条件下，醉马草-内生真菌共生体生物碱粗提液显示了良好的杀虫或者驱虫效果，初步的研究结果表明，试验小区的喷洒量达到200 mL/10m2时，醉马草提取液可以有效缓解朱砂叶螨和禾谷缢管蚜对寄主植物的危害。自醉马草植株提取的挥发油和生物碱提取液均对8种禾本科植物病原真菌的菌落生长、孢子萌发和芽管伸长均具有显著的抑制作用，其中生物碱对腐皮镰孢菌落生长、孢子萌发和芽管伸长的抑制率最大，分别达到63%、57%和68%。生物碱浓度与供试病原真菌孢子萌发抑制率和芽管伸长均呈显著正相关(P<0.05)。两种麦角生物碱对动物平滑肌细胞均具有明显的细胞毒活性，且生物碱浓度与细胞的抑制率呈显著正相关(P<0.05)，回归方程分别为：Y麦角=0.0041x＋0.205(R2=0.945，P<0.05)和Y异麦角=0.0032x＋0.2672 (R2=0.9411，P<0.05)，麦角新碱和异麦角新碱的半致死浓度分别为71.95 µg/mL和72.75 µg/mL。