中国科学院海洋研究所机构知识库

  植物病害每年都给农作物的生产造成巨大损失，植物病原真菌则是导致植物病害的主要原因之一。长期以来，人们一直使用化学农药防治植物病害。其中，金属农药因其具有杀菌性好、成本低、不产生抗药性等优点而被广泛使用。但是，大量金属农药的使用也会产生药害、重金属污染等问题。因此，以传统化学金属农药为基础，设计合成天然有机金属农药，是新型金属农药研发的主要方向。壳聚糖是一种天然高分子化合物，它无毒无害且资源丰富，本身就具有抑菌杀菌作用，但因其效果不强、水溶性差，应用受到了限制。将壳聚糖进行衍生化进而与金属离子相结合，有望制备出活性高、无药害、绿色、安全的农用海洋生物金属制剂，解决现有金属制剂存在的问题。

  本文以海洋生物中提取的壳聚糖为原料，分别通过席夫碱化以及金属络合等方式，对壳聚糖进行化学改性。共合成了14种壳聚糖席夫碱和24种壳聚糖席夫碱金属复合物。采用红外光谱、核磁共振波谱、元素分析、差示扫描量热、X射线衍射等手段对衍生物的理化性质进行了全面解析。同时，还使用了新兴的密度泛函理论，结合计算化学对壳聚糖衍生物的分子构象进行了研究。通过体外抑菌活性实验，探究了壳聚糖衍生物的抑菌活性和杀菌谱，并筛选出4种衍生物进行了盆栽实验，18种衍生物进行了植物毒性实验和细胞毒性实验。主要研究结果如下：

1. 制备了6种壳聚糖氨基苯甲酸席夫碱及6种壳聚糖铜复合物。首次使用密度泛函理论解析了壳聚糖铜复合物的单元结构及构型。抑菌结果表明，合成的12种壳聚糖衍生物的抑菌活性均好于壳聚糖以及阳性对照噻菌铜，衍生物中的空间位阻对抑菌效果有影响，结构单元中p-π共轭与苯环的二面角越大，抑菌效果越好。
2. 制备了2种壳聚糖烟酰胺类席夫碱及6种壳聚糖金属复合物。抑菌实验表明：6种壳聚糖金属复合物的抑菌效果均强于壳聚糖，使用烟酰胺修饰的金属复合物的抑菌效果好于使用异烟酰胺修饰的金属复合物。不同金属离子络合后的复合物对不同病原真菌也表现出不同的抑菌活性。其中，铜复合物对辣椒疫霉病菌的抑制效果最好，镍复合物对灰葡萄孢病菌的抑制效果最好。植物安全性实验表明：6种金属复合物对小麦叶片的毒性均小于游离金属离子，对植物生长没有显著影响。其中，壳聚糖锌复合物还能提高小麦叶片中的叶绿素含量。
3. 制备了4种壳聚糖氨基吡啶席夫碱及12种壳聚糖金属复合物。体外抑菌结果表明：12种金属复合物具有很好的抑菌效果和广谱杀菌活性。铜复合物对辣椒疫霉病菌和黑白轮枝孢病菌抑制效果较好，镍复合物对灰葡萄孢病菌抑制效果较好。盆栽实验表明，复合物的抑菌活性与氨基吡啶上的取代基种类有关，其中，甲基效果最好。

  本论文制备并筛选出6种结构新颖、抑菌活性较好的壳聚糖衍生物，对其植物毒性等特性进行了研究。衍生物基本保留了壳聚糖毒性低、生物相容性好等优点。而且，上述衍生物的抑菌活性大多好于市售农药噻菌铜、好普寡糖等产品，这为新型农用杀菌剂的研发提供了一个新的方向。同时，文中所使用的计算化学方法也为多糖等大分子的结构解析提供了一个新的思路。

  Plant diseases cause considerable losses to crop yields every year. Plant pathogenic fungi are one of the main causes of plant diseases. Chemical pesticides have been used to control plant diseases for a long time. Among them, metal pesticides are widely used because of their advantages such as low cost, broad antimicrobial spectrum and strong sterilization effect. However, the use of metal fungicides will also cause problems such as phytotoxicity and heavy metal contamination. Therefore, the design and synthesis of natural organic metal pesticides based on traditional chemical metal pesticides is the main direction of research and development of metal pesticides. Chitosan is a natural macromolecule compound. It is innoxious and harmless and is rich in resources. Chitosan has antifungal effects, but its application is limited because of its poor effect and poor water solubility. Hence, we expect that grafting Schiff base and metal ions onto chitosan may result in the development of a new metal complex with a novel character and enhanced antifungal activity, so as to solve the problems of traditional metal fungicides.

  In this paper, the chitosan obtained from marine organisms was chemically modified by Schiff base and metal complexation respectively. Fourteen kinds of chitosan Schiff bases and twenty-four kinds of chitosan Schiff base complexes were synthesized. The physical and chemical properties of the derivatives were comprehensively analyzed by infrared spectroscopy (FTIR), nuclear magnetic resonance spectroscopy (NMR), elemental analysis, differential scanning calorimetry (DSC) and X-ray diffraction (XRD). At the same time, the new density functional theory (DFT) was used, and the molecular conformations of chitosan derivatives were calculated with the combination of computational chemistry. The bactericidal spectrum of chitosan derivatives was investigated through the antifungal activity experiments in vitro. Four kinds of derivatives were screened out for pot experiments, 18 derivatives were tested for phytotoxicity and cytotoxicity. The main results were as the followings:

1. Six kinds of chitosan aminobenzoic acid Schiff base and six kinds of chitosan aminobenzoic acid Schiff base copper complexes were prepared. The unit structure and configuration of chitosan copper complex were analyzed by density functional theory (DFT) for the first time. The antifungal results showed that the antifungal activity of twelve kinds of chitosan derivatives are better than that of chitosan and thiediazole copper. The inhibition of chitosan copper complex against Phytophthora capsici has a positive correlation with the dihedral angle between the phenyl and carbon-nitrogen p-π conjugate groups in chitosan copper complex.
2. Two kinds of chitosan nicotinamide Schiff base and six kinds of chitosan nicotinamide Schiff base metal complexes were prepared. Fungistatic experiments showed that the antifungal effects of the six chitosan metal complexes were better than that of chitosan, and the antifungal effect of nicotinamide modified metal complexes was better than that of metal complexes modified with isonicotinamide. The complex of different metal ions also showed different fungistasis to different pathogenic fungi. The inhibitory effect of copper complex against Phytophthora capsici was the best, and the nickel complex had the best inhibition effect against Botrytis cinerea. The phytotoxicity assay showed that the toxicity of six kinds of metal complexes on wheat leaves was less than that of free metal ions and had no significant effect on plant growth. Moreover, the content of chlorophyll in wheat leaves could be improved by the zinc complexes.
3. Four kinds of chitosan aminopyridine Schiff base and twelve kinds of chitosan aminopyridine Schiff base metal complexes were prepared. The results of fungistasis in vitro showed that the twelve kinds of metal complexes had good fungistasis effect and broad-spectrum fungicidal effect. The inhibition effect of copper complexes on Phytophthora capsici and Verticillium alboatrum was better, and the inhibitory effect of nickel complex on the Botrytis cinerea was better. Pot experiments showed that the antifungal activity of the complexes was related to the kinds of substituents on aminopyridine. Methyl group was the best.

  In this paper, six kinds of chitosan derivatives with novel structure and good antifungal activity were prepared and screened, and their phytotoxicity and other characteristics were studied. The derivatives basically retain the advantages of chitosan, such as low toxicity and good biocompatibility. Moreover, the antifungal activity of the derivatives is better than that of thiediazole copper and oligosaccharides. The experimental results may provide a novel direction for the development of metal fungicides, and the characterization of new derivatives provides a novel research idea in structural analysis.

摘 要... I

ABSTRACT. III

目  录... VII

第一章 绪论... 1

第一节 农用金属杀菌剂的应用现状及发展趋势... 1

1.1 金属杀菌剂简介... 2

1.2 几种金属杀菌剂的概况... 4

1.3 金属杀菌剂的发展趋势... 7

第二节 甲壳素与壳聚糖... 8

2.1 甲壳素、壳聚糖简介... 8

2.2 壳聚糖的应用现状... 10

第三节 壳聚糖金属复合物的抑菌活性研究进展... 12

第四节 选题意义与研究内容... 15

第二章 壳聚糖金属复合物的制备及结构表征... 17

第一节 壳聚糖氨基苯甲酸席夫碱铜复合物的制备及结构表征... 17

1 原料与仪器... 17

2 实验方法... 18

3 结果与讨论... 23

第二节 壳聚糖烟酰胺类席夫碱金属复合物的制备及结构表征... 37

1 原料与仪器... 37

2 实验方法... 38

3 结果与讨论... 41

第三节 壳聚糖氨基吡啶席夫碱金属复合物的制备及结构表征... 52

1 原料与仪器... 52

2 实验方法... 53

3 结果与讨论... 56

本章小结... 69

第三章 壳聚糖金属复合物的抑菌活性测定... 70

第一节 壳聚糖氨基苯甲酸席夫碱铜复合物的抑菌活性测定... 70

1 原料与仪器... 70

2 实验方法... 71

3 结果与讨论... 72

第二节 壳聚糖烟酰胺类席夫碱金属复合物的抑菌活性测定... 79

1 原料与仪器... 79

2 实验方法... 79

3 结果与讨论... 80

第三节 壳聚糖氨基吡啶席夫碱金属复合物的抑菌活性测定... 88

1 原料与仪器... 88

2 实验方法... 89

3 结果与讨论... 91

本章小结... 99

第四章 壳聚糖金属复合物的生物安全性研究... 101

第一节 壳聚糖金属复合物的植物毒性研究... 101

1 原料与仪器... 101

2 实验方法... 102

3 结果与讨论... 103

第二节 壳聚糖金属复合物的动物细胞毒性研究... 108

1 原料与仪器... 108

2 实验方法... 109

3 结果与讨论... 109

本章小结... 112

第五章 结论和创新点... 113

本文结论... 113

创新点... 116

参考文献... 117

附  录... 129

致  谢... 131

个人简历及攻读学位期间发表的学术论文与研究成果    132