紫茎泽兰甲醇提取物对石膏样小孢子菌的抑制作用及其机制

Inhibition effect of Ageratina adenophorais methanol extracts against Microsporidium gypsum and its mechanism

通过硅胶柱层析、薄层层析和气相色谱-质谱联用方法,从紫茎泽兰( Ageratina adenophorais )甲醇提取物中分离、鉴定出3类化合物,分别为9-羰基-10,11-去氢泽兰酮(euptox A)、3-烯-2,7-二酮-杜松萜烯(CED)和烯烃类化合物。使用含毒介质法分别检测125、62.5、31.25、15.6、7.8、3.9、1.95和0.98 μg ·mL -1 的euptox A和CED在48、72、96、120、144 h时对石膏样小孢子菌( Microsporidium gypsum )的抑制率。对euptox A处理过的石膏样小孢子菌开展碘化丙啶(PI)染色试验,并测定其细胞膜麦角固醇含量,以及细胞外液中总蛋白质和总磷的含量。结果显示,紫茎泽兰甲醇提取物中euptox A和CED具有一定的抗真菌活性,且以euptox A的效果更好。经euptox A作用后,石膏样小孢子菌的菌丝在荧光显微镜下呈现出强红色荧光,细胞外液中总蛋白质和总磷含量显著( P <0.05)增加,麦角固醇的含量显著( P <0.05)降低。据此推测,euptox A作用于石膏样小孢子菌后,可通过抑制其麦角固醇合成,使细胞膜完整性受到破坏,导致细胞内外物质交换紊乱,从而最终引起真菌细胞死亡。

In the present study,9-oxo-10,11-dehydroagerophorone (euptox A),cadinan-3-ene-2,7-dione (CED) and olefin compounds were separated and identified from Ageratina adenophorais methanol extract by silica gel column chromatography,thin-layer-chromatography and gas chromatography-mass spectrometer. The inhibition effects of125,62.5,31.25,15.6,7.8,3.9,1.95 and 0.98 μg ·mL -1 euptox A and CED against Microsporidium gypsum were determined at 48,72,96,120,144 h,respectively. The Microsporidium gypsum samples treated with euptox A were selected as test materials,dye penetration test with propidium iodide (PI) was carried out,and the content of total protein and total phosphorus in extra-cellular fluid,and ergo-sterol content in mycelium were determined. It was shown that the separated euptox A and CED from Ageratina adenophorais methanol extract could inhibit the growth of Microsporidium gypsum, and euptox A possessed stronger inhibition effect. After the treatment with euptox A,the hyphae of M icrosporidium gypsum showed strong red fluorescence under fluorescence microscope. The contents of total protein and total phosphorus in extracellular fluid of Microsporidium gypsum were significantly ( P <0.05) increased,yet the ergo-sterol content in mycelium was significantly ( P <0.05) decreased. It was inferred that euptox A could inhibit the synthesis of ergo-sterol,cause membrane damage,and lead to cellular disorder and cell death.