【作者】 王娟；

【导师】 高锦明；

【作者基本信息】 西北农林科技大学 ， 植物学， 2007， 硕士

【摘要】 本文以微生物发酵学、植物化学和现代化学分离理论为基础,在对两种高等真菌硫磺菌和灰盖鬼伞发酵液及菌丝体提取物进行抑菌杀虫活性检测的前提下,对活性较好的灰盖鬼伞菌丝体及发酵液乙酸乙酯萃取物的化学成分进行了分离纯化和鉴定,并对纯化合物进行了抑菌活性测定。研究结果如下:采用药敏纸片法测定了硫磺菌发酵液乙酸乙酯和正丁醇萃取物的抑菌活性。试验结果表明,不同浓度的硫磺菌发酵液乙酸乙酯萃取物对7种供试细菌、8种植物病原菌和黑根霉均有明显的抑菌活性。除对大肠杆菌、金黄色葡萄球菌、黑根霉和棉花黄萎病菌外,均表现为浓度越高抑菌活性越强的趋势;而不同浓度的正丁醇萃取物对3种细菌、3种霉菌和5种植物病原菌也具有较明显的抑菌作用,除对黑曲霉和苹果褐腐病菌外,随着浓度的增大抑菌活性也明显增强。而不同浓度的处理对马铃薯芽孢杆菌的抑菌活性相当。采用药敏纸片法和抑制菌丝生长速率法测定了灰盖鬼伞菌丝体和发酵液的乙酸乙酯萃取物和正丁醇萃取物的抑菌活性。试验结果表明,不同浓度的菌丝体乙酸乙酯萃取物对9种细菌和9种植物病原菌均有较明显的抑菌活性。除对灵杆菌和葡萄炭疽病菌外,均随着浓度的增加抑菌活性增强,而对葡萄炭疽病菌则表现为“低抑高促”的现象;不同浓度的菌丝体正丁醇萃取物对8种细菌均有一定的抑制作用,但作用没有乙酸乙酯萃取物强,而对10种病原菌的抑制作用却较之后者要好;不同浓度的发酵液乙酸乙酯和正丁醇萃取物对10种病原菌均具有较明显的活性。采用叶碟法测定了硫磺菌和灰盖鬼伞发酵产物对3龄粘虫的拒食和毒杀活性。结果发现在10mg/mL的供试浓度下,硫磺菌和灰盖鬼伞菌丝体和发酵液甲醇提取物以及乙酸乙酯和正丁醇萃取物的拒食活性相当,且都不明显。而灰盖鬼伞发酵产物的毒杀活性较之硫磺菌发酵产物的要强得多,其菌丝体和发酵液乙酸乙酯萃取物活性较好,第3d其校正死亡率都超过50%,到第4d校正死亡率都达到70%以上。本文分别对灰盖鬼伞菌丝体和发酵液的乙酸乙酯萃取物采用常压硅胶柱层析和反相硅胶柱层析分离、Sephadex LH-20葡聚糖凝胶柱纯化,最终分到W-1、W-2、W-3、W-4、W-5、W-6、W-7共7个化合物,其中W-4因易被氧化未能做波谱鉴定, W-1、W-2、W-3、W-7经理化性质和有机波谱鉴定(MS、1H NMR、13C NMR和DEPT),以及经与标准化合物对照,确定出其结构分别为麦角甾-4,6,8(14),22-四烯-3-酮[ergosta-4,6,8(14),22-tetraen-3-one]、麦角甾-5, 22-二烯- 3β-醇[(22E,24R)-ergosta-6, 22- dien- 3β- ol]、过氧化麦角甾醇[5α,8α-epidioxy-(22E,24R)-ergosta-6,22-dien-3β-ol]和一大环内酯类化合物[ (E)- 2- hexl-2,3,4,4a,7,8,9,10-octahydro-9,10,12a-trihydroxypyrano-[3,2-b] oxecin-6(12aH)-one],其中W-2、W-3和W-7首次在灰盖鬼伞发酵产物中发现。另外,对经过常压硅胶柱层析分离后的灰盖鬼伞菌丝体J1和J2部分、发酵液的F1部分进行了GC-MS分析。分析结果表明灰盖鬼伞菌丝体中含有6种脂肪酸,以不饱和脂肪酸的含量最高,占总脂肪酸的93.35%,其次为棕榈酸(占13.84%),硬脂酸(占2.81%);发酵液中含有5种脂肪酸,也是不饱和脂肪酸的含量最高,占总脂肪酸的64.99%,其次为棕榈酸(占25.30%)和硬脂酸(占9.67%)。在浓度为5mg/mL时,已鉴定出结构的4个单体化合物对5种细菌均有不同程度的抑制作用。其中,W-2和W-7对金黄色葡萄球菌、枯草芽孢杆菌、变形杆菌、灵杆菌和大肠杆菌的抑菌活性较好,而W-1和W-3仅对金黄色葡萄球菌具有一定的抑制作用,对其它4种细菌的活性都不显著。更多还原

【Abstract】 This thesis, guided by microbial zymology, phytochemistry and theory of chemical separation, deals with the antimicrobial and insecticidal activity of extracts from fermented liquor and mycelia of two higher fungi Laetiporus sulphureus and Coprinus cinereus in the elementary stage, then deals with the isolation, purification and determination of chemical components of Ethyl acetate extracts from fermented liquor and mycelia of Coprinus cinereus which showed better bioactivity than Laetiporus sulphureus did.The following are the results of the study.The fermented broth of Laetiporus sulphureus was tested by Neo-Sensitab disk-diffusion method for antimicrobial activity. The results showed that Ethyl acetate extract with different concentration from the fermented broth had noticeable antimicrobial activity against 7 kinds of tested bacteria, Rhizopus nigricans and 8 kinds of plant pathogens,and the higher concentration,the stronger antimicrobial was,excepting against Escherichia coli, Staphylococcus aureus, Rhizopus nigricans and Verticillicum dahliae.while the n-Butanol extract alse exhibited significant activity on 3 kinds of bacteria,3 kinds of moulds and 5 kinds of pathogens.with the increaseing of tested concentration,the antimicrobial activity increased except on Aspergillus niger,Monilia fructigena, but the inhibitory effects kept the same on Bacillus mesentericus.The fermented mycelia and broth of Coprinus cinereus were tested f by Neo-Sensitab disk-diffusion method and Mycelia Grow Inhibition method for antimicrobial activity. The results showed that Ethyl acetate extract with different concentration from mycelia had obvious antimicrobial activity against 9 kinds of bacteria, 9 kinds of plant pathogens, and the higher concentration, the stronger antimicrobial was, excepting on Bacterium prodigious and Colletotrichum gloeosporioides.While the n-Butanol extract with different concentration exhibited better activity on 10 kinds of pathogens, weaker activity on 8 kinds of bacteria than the Ethyl acetate extract did. The Ethyl acetate and n-Butanol extracts with different concentration from fermented broth exhibited certain activity against 10 kinds of plant pathogens.The antifeeding and insecticidal activities of different polar solvent extracts from fermented mycelia and broth were tested by Leaf Disc method. Results showed that the fermentation extracts of Laetiporus sulphureus and Coprinus cinereus showed the same antifeeding activities against the third instar larvae of Mythimna separata, and both the antifeeding rate displayed weak. The Ethyl acetate extracts from the fermenthing broth and mycelia of Coprinus cinereus displayed better toxic activities than that of Laetiporus sulphureus did, the morality rate of which in 3d surpassed 50% and 4d surpassed 70% on Mythimna separata.Seven compounds were isolated from the Ethyl acetate extracts of mycelia and fermented broth of Coprinus cinereus by means of silica gel and reverse phase(RP-18) silica-gel colum chromatography,and were further purified by Sephedex LH-20.Among them four compounds were identified.Compound W-1’s structure was identified by direct comparison with authentic sample as ergosta-4,6,8(14),22-tetraen-3-one.the other compounds’structures were elucidated on the analysis of their spectral data(MS,1H-NMR,13C-NMR and DEPT) and compared with the references.They were ergosta-5,22-dien-3β-ol(W-2), 5α,8α-epidioxy-(22E,24R)-ergosta-6,22-dien-3β-ol(W-3) and a new macrolactin ( (E)- 2- hexl -2,3,4,4a,7,8,9,10-octahydro-9,10,12a-trihydroxypyrano-[3,2-b]oxecin-6(12aH)-one ) (W-7), in which compounds W-2, W-3 and W-7 were isolated from Coprinus cinereus firstly.Fraction J1, J2 and F1 which were isolated from the Ethyl acetate extracts, were analyzed by GC-MS, The result showed that six fatty acids were defined from mycelia, in which contained unsaturated fatty acids (93.35%), palmitic acid (13.84%), stearic acid (2.81%).five fatty acids were defined from fermented broth, in which contained unsaturated fatty acids (64.99%), palmitic acid (25.30%) and stearic acid (9.67%).Four identified compounds had some inhibition activity against five kinds of bacteria, in which compounds W-2 and W-7 behaved obvious inhibition effect on tested bacteria, but compounds W-1 and W-3 had no effect excepting on Staphylococcus aureus.更多还原