摘要
采用酒精和乙酸乙酯分级溶解、结晶方法快速简便地纯化到97.5％的蛇床子素(Osthol)，用过氧乙酸对蛇床子素进行氧化修饰得到两个产物，这两个产物对小麦赤霉菌的抑菌活性与蛇床子素相比没有显著差异。
离体实验表明，蛇床子素对苹果叶枯病菌、苹果轮纹病菌等植物病原真菌有着较广谱的抑制活性。研究结果表明蛇床子素抑制小麦赤霉菌丝顶端生长，使其分枝增多；同时对小麦赤霉菌孢子萌发有显著抑制作用，当其浓度达到50 μ g·mL~(-1)，小麦赤霉菌孢子萌发被完全抑制。
液体培养条件下，以100 μ g·mL~(-1)蛇床子素处理小麦赤霉菌24h，造成菌丝大量断裂。对细胞壁形成所需底物和细胞壁水解相关酶测定，结果表明蛇床子素处理后小麦赤霉菌体内几丁质酶活性、N-乙酰葡萄糖胺含量均升高，而β-1.6-葡聚糖酶活性与对照相比无显著变化。在培养基中加入~(14)C标记葡萄糖，以100 μ g·mL~(-1)蛇床子素处理小麦赤霉菌，结果表明菌体内葡萄糖含量迅速下降，3h时达到最低，为对照51％，同时几丁质酶活性迅速升高，3h时为对照3倍；处理后可溶性蛋白质和蛋白酶活性均升高；由此推测糖饥饿诱导几丁质水解酶表达，引起几丁质前体水解，细胞壁形成受阻。
以100 μ g·mL~(-1)蛇床子素处理小麦赤霉菌后12h，处理菌体内钙含量仅为对照的60％，表明蛇床子素能抑制菌体对钙的吸收，推测其影响到Ca~(2+)依赖泡囊内含物运输释放到细胞壁过程，从而影响到菌丝顶端生长。
此外，用PAGE法研究发现蛇床子素还能体外抑制小麦赤霉菌ATPase活性，表明其机制还在于通过抑制能量系统而起作用。
97.5% purity of osthol was achieved by gradient resolvement and crystallization with ethanol and ethyl acetate. When osthol was treated with Acidum Peroxyaceticum, two main products were achieved, but the antifungal activity of the two products did not increase compared with osthol.
In vitro studies,osthol displayed a wide range of antifungal activity to phytopathogenic fungi such as Rhizoctonia solani and Macrophoma kawatsukai. Osthol inhibited hyphal growth and spores germination of F.graminearuym. At 50 g mL-1 osthol,Spores germination was completely inhibited and hyphal branches increased. Treated with 100 g mL-1osthol for 24 hour, the hyphae growing in aliquous culture fragmented greatly.
In 100 g mL-1 osthol-treated F.graminearuym, N-GlcNAc required for synthesis of cell wall as well as cell wall lysis-related chitinase activity increased whereas -1.6-glucanase activity remain unchanged. When F. graminearuym fed with 14C glucose was treated with 100 g mL-1 osthol, glucose content decreased rapidly and reduced to the lowest level i.e. 51% of control in 3h, while chitinase activity,soluable proteins content and protease activity increased rapidly,which suggested glucose starvation might induce the expression of chitinase. It was proposed that osthol treatment increased chitinase activity leading to the degradation of chitinsome required for biosynthesis of fungus cell wall and led to hyphae fragment.
When F. gramineantymg was treated with 100 g mL-1 osthol for 12h, calcium in F. graminearuym decreased by 40%. Such suggesteded that osthol inhibited uptake of calcium required for Ca2+ dependent vacuole transportation and growing hyphae tip. PAGE indicated that osthol also inhibited ATPase, and it was likely that osthol could inhibit energy metabolism by inhibiting ATPase.
引文
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