学位论文
学位类型硕士
导师季乃云
2019-05-16
学位授予单位中国科学院大学
学位授予地点北京
学位名称工程硕士
学位专业生物工程
关键词海洋来源真菌次生代谢产物化学结构生物活性
摘要? 海洋占地球表面积的三分之二以上，地球上近五分之四的生命在海洋中繁衍，海洋是具有丰富资源的宝库。海洋特殊的生态环境使得海洋生物更加的耐盐、耐饥和耐压等，因而海洋真菌的代谢途径与陆生真菌的代谢途径有很大的差异，其产生的次生代谢产物的结构更加特殊多样，生物活性和药理性质更加强烈。因此越来越多的研究者将新型药物开发的希望寄于海洋真菌次生代谢产物的发现上。
?本文分别选取了3株不同来源的海洋真菌并对其次生代谢产物进行研究，分别为来自威海海域冈村凹顶藻的长枝木霉（Trichoderma longibrachiatum）A-WH-20-2、来自大连海域松节藻的长枝木霉（T. longibrachiatum）RR-DL5-4和来自烟台海域沉积物中的阿姆斯特丹曲霉（Aspergillus amstelodami）3-7。从上述3株真菌中共分离得到49个次生代谢产物，包含了萜类、甾体类、哌嗪类、吲哚类和内酯类等结构类型，并对所分离的化合物进行了抗细菌、抗真菌、抗微藻和卤虫毒性的生物活性评价。
? 用有机溶剂提取法将样品中的化合物反复浸提出来。采用柱色谱法（硅胶柱层析、反相柱层析、Sephadex LH-20凝胶柱层析），薄层色谱法（制备型薄层色谱）与高效液相色谱法，利用常规分离方法与现代仪器分离方法相结合的方式来获得单体化合物。采用比对文献中数据和波谱法（核磁共振谱、质谱、红外光谱、紫外光谱等方法）来确定单体化合物的结构。从长枝木霉A-WH-20-2中共得到26个单体化合物，包括1个新化合物A1和1个新天然产物A2。从长枝木霉RR-DL5-4中共分离得到18个单体化合物，两株长枝木霉代谢产物有很大的相似性。从阿姆斯特丹曲霉中共分离得到5个单体化合物。
? 将所得到的化合物进行了抗菌、抗微藻和卤虫毒性的活性试验。活性测试表明，化合物A5、A6和B3对真菌围小丛壳有显著的抑制活性，活性优于阳性对照两性霉素B。化合物A19对枯草芽孢杆菌有较好的抑制活性。化合物A1–A8、A13、A17、A19–A21、A23、A26、B3、C2、C3和C5在终浓度100 μg/mL时对赤潮异弯藻、东海原甲藻、剧毒卡尔藻和海洋卡盾藻的抑制率达到100%。
? 本文系统的研究了3株海洋真菌次生代谢产物的化学结构和生物活性，不仅丰富了海洋真菌次生代谢产物的研究内容，还发现了一些具有活性的单体化合物，为海洋真菌资源的进一步开发利用提供了一定的科学依据。?
其他摘要? The sea covers more than two-thirds of the earth's surface, and nearly four-fifths of all lives on earth thrive in the oceans. So, the sea is an enormous treasure trove of resources. The harsh ecological environment of the ocean makes marine organisms more tolerant to salt, hunger, and pressure. Therefore, the metabolic pathways of marine fungi are quite different from those of terrestrial fungi. The secondary metabolites produced by marine-derived fungi have more special and diverse structures and stronger biological activity and pharmacological properties. Hence, more and more researchers pin their hope for new drug development on the discovery of secondary metabolites.
? In this paper, three marine-derived fungi from different sources were selected as objects. Trichoderma longibrachiatum A-WH-20-2 was isolated from the marine alga Laurencia okamurai collected from the coasts of Weihai. T. longibrachiatum RR-DL5-4 was isolated from the marine alga Rhodomela confervoides collected from the coasts of Dalian. Aspergillus amstelodami 3-7 was isolated from the marine sediments collected from Yantai coasts. Totally, forty-nine secondary metabolites, including steroids, phenols, terpenes, piperazines, and lactones, were isolated from the three fungal strains, and their bioactivities against bacteria, fungi, microalgae, and brine shrimp were evaluated.
? The compounds in the sample were repeatedly extracted by organic solvents. Pure compounds were obtained by column chromatography (silica gel column chromatography, RP-18 reversed-phase silica gel, Sephadex LH-20 gel column chromatography), thin-layer chromatography (TLC), and high performance liquid chromatography (HPLC). A total of 26 compounds, including a new compound A1 and a new naturally occurring compond A2, were obtained from T. longibrachiatum A-WH-20-2. 18 compounds were isolated from T. longibrachiatum RR-DL5-4. The metabolites of the two strains were very similar. Additionally, 5 compounds were isolated from A. amstelodami.
? All the compounds were tested for antibacterial, antifungal, and antimicroalgal activities as well as brine shrimp lethality. The results showed that compounds A5, A6, and B3 showed significant inhibitory activity against Glomerella cingulata, and the activities were better than that of amphotericin B. Compound A19 has good inhibitory activity against Bacillus subtilis. The inhibition rates of compounds A1–A8, A13, A17, A19–A21, A23, A26, B3, C2, C3, and C5 on Heterosigma akashiwo, Chattonella marina, Prorocentrum donghaiense, and Karlodinium veneficum reached 100% at the final concentration of 100 μg/mL.
? In this paper, the secondary metabolites of three marine-derived fungi and their biological activities were systematically studied. This not only enriched the research content of secondary metabolites of marine-derived fungi, but also found some active compounds. This study contributes to the further development and utilization of marine-derived fungi.
目录目 录
第1章 引言................................................................................................................. 1
1.1 近五年海洋真菌次生代谢产物研究概况............................................................ 2
1.1.1 海藻来源真菌................................................................................................. 2
1.1.2 动物来源真菌................................................................................................. 9
1.1.3 沉积物（包括深海）来源真菌................................................................... 21
1.1.4 红树林来源真菌........................................................................................... 27
1.2 本论文研究意义.................................................................................................. 35
第2章 长枝木霉A-WH-20-2次生代谢产物的分离纯化与结构鉴定.................. 37
2.1 化合物结构鉴定.................................................................................................. 37
2.1.1 新化合物结构鉴定....................................................................................... 37
2.1.2 已知化合物结构鉴定................................................................................... 40
2.2 实验部分.............................................................................................................. 48
2.2.1 仪器与试剂................................................................................................... 48
2.2.2菌种鉴定........................................................................................................ 49
2.2.3 菌株发酵....................................................................................................... 49
2.2.4 提取与分离纯化........................................................................................... 49
2.2.5 化合物物理常数和波谱数据....................................................................... 50
2.3 小结...................................................................................................................... 59
第3章 长枝木霉RR-DL5-4次生代谢产物的分离纯化与结构鉴定.................... 61
3.1 化合物的结构鉴定.............................................................................................. 61
3.1.1 已知化合物结构鉴定................................................................................... 61
3.2 实验部分.............................................................................................................. 65
3.2.1 仪器与试剂................................................................................................... 65
3.2.2 菌种鉴定....................................................................................................... 65
3.2.3 长枝木霉RR-DL5-4的发酵条件筛选及规模发酵培养............................ 66
3.2.4 提取与分离纯化........................................................................................... 66
3.2.5 化合物波谱数据........................................................................................... 67
3.3 小结...................................................................................................................... 70
第4章 阿姆斯特丹曲霉3-7次生代谢产物的分离纯化与结构鉴定.................... 71
4.1 化合物结构鉴定.................................................................................................. 71
4.1.1 已知化合物结构鉴定................................................................................... 71
4.2 实验部分.............................................................................................................. 73
4.2.1 仪器与试剂................................................................................................... 73
4.2.2 菌种鉴定....................................................................................................... 73
4.2.3 菌株发酵....................................................................................................... 73
4.2.4 提取与分离纯化........................................................................................... 73
4.2.5 化合物波谱常数........................................................................................... 74
4.3 小结...................................................................................................................... 76
第5章 单体化合物活性测试................................................................................... 77
5.1 活性测试材料...................................................................................................... 77
5.2 活性测试方法...................................................................................................... 77
5.3 活性测试结果...................................................................................................... 78
5.3.1 抑菌活性测试结果....................................................................................... 78
5.3.2 抗微藻活性测试结果................................................................................... 79
5.3.3 盐水丰年虾毒性测试结果........................................................................... 81
5.3 小结...................................................................................................................... 82
第6章 结语............................................................................................................... 83
6.1 结论...................................................................................................................... 83
6.2 创新点.................................................................................................................. 83
参考文献..................................................................................................................... 85
附 录........................................................................................................................... 99
致 谢......................................................................................................................... 107
作者简历及攻读学位期间发表的学术论文与研究成果....................................... 109
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页数110
语种中文
文献类型学位论文
条目标识符http://ir.yic.ac.cnhttp://ir.yic.ac.cn/handle/133337/25296
专题中国科学院烟台海岸带研究所
推荐引用方式
GB/T 7714学位论文. 三株海洋真菌次生代谢产物及其生物活性研究[D]. 北京. 中国科学院大学,2019.
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