海岸带生物学与生物资源保护实验室知识库

甲壳素是自然界中来源广泛、储存量丰富的一种天然含氮多糖，具有良好的生物相容性、生物可降解性等特性，甲壳素经脱乙酰得到壳聚糖。壳聚糖是自然界中唯一的一种天然碱性多糖，具有无毒性、成膜性、吸湿保湿性、抗氧化、抑菌等独特的生物活性，但是由于其分子内及分子间的氢键使分子的刚性结构增强，导致壳聚糖在水或者碱性溶液中溶解性较差，另外，壳聚糖本身的抑菌和抗氧化活性较弱，不能满足高效地发展需求。化学改性是改善壳聚糖水溶性、提高其生物活性的有效手段之一，如酰基化、季铵化、磷酸化等修饰方法，其中，季铵化改性产物由于具有良好的水溶性和高的生物活性而受到广泛关注。

本文综合采用了制备壳聚糖季铵盐的两种途径，在壳聚糖季铵盐的基础上，进行季铵化再修饰，合成了一系列含有不同季铵盐数目、不同活性官能团的壳聚糖季铵盐的衍生物，利用红外、核磁共振和元素分析等手段对产物的结构进行表征，通过清除超氧阴离子自由基、DPPH自由基和羟基自由基测试壳聚糖季铵盐的衍生物的抗氧化活性，利用生长速率抑制法测定壳聚糖季铵盐的衍生物对灰葡萄孢菌、芦笋茎枯致病菌、玉蜀黍赤霉、梨轮纹病菌、黄瓜枯萎致病菌及西瓜枯萎致病菌等几种常见植物致病菌的抑菌能力大小，从而筛选出具有高抗氧化活性和高抑菌活性的壳聚糖衍生物。

氨基多糖往往具有特殊的生理活性，例如抗氧化、抑菌、抗肿瘤、抗凝血等。在本论文中，首先对壳聚糖2位氨基烷基化得到N,N,N-三甲基壳聚糖衍生物（TMC），不仅增加了壳聚糖的正电荷密度，同时也增加了产物的溶解性，利于反应的进一步进行。在TMC的基础上，利用含氮亲核试剂分别将氨乙基以O和N两种不同的连接方式引入到壳聚糖6位羟基位置，得到了N,N,N-三甲基-6-O/N-氨乙基壳聚糖季铵盐的衍生物，并对其抗氧化活性和抑菌活性进行了测试。抗氧化结果表明，在1.6 mg/mL 样品浓度下，与壳聚糖相比，N,N,N-三甲基-6-N-氨乙基壳聚糖衍生物和N,N,N-三甲基-6-O-氨乙基壳聚糖衍生物对超氧阴离子自由基和DPPH自由基的清除率均提高了40.0%左右，在羟基自由基清除过程中，N,N,N-三甲基-6-N-氨乙基壳聚糖衍生物对其清除率大于90.0%。与TMC相比，两种产物的抗氧化活性降低了。抑菌活性测试结果表明，与壳聚糖相比，N,N,N-三甲基-6-O/N-氨乙基壳聚糖季铵盐的衍生物抑菌活性显著增强，例如在1.0 mg/mL 浓度下，N,N,N-三甲基-6-N-氨乙基壳聚糖衍生物对灰葡萄孢菌的抑菌率可以达到90.0%。另外，与TMC相比，N,N,N-三甲基-6-O/N-氨乙基壳聚糖季铵盐的衍生物的抑菌活性得到进一步增强。

在壳聚糖衍生物中，杂环结构往往具有良好的生物活性，例如含N杂环吡啶因其良好的抑菌性而被广泛应用到农药领域。在本论文中，通过希夫碱反应，将吡啶环引入到壳聚糖分子中，利用吡啶环上N原子的三级胺性质，对其进行烷基化，得到含有吡啶盐的壳聚糖双季铵盐衍生物。研究结果表明壳聚糖双季铵盐衍生物的抗氧化活性得到增强，特别是在样品浓度为1.6 mg/mL 时，壳聚糖双季铵盐衍生物清除DPPH自由基的能力可以与Vc的媲美，同时，对羟基自由基也有较好的清除能力，特别是吡啶环上3位取代产物几乎可以完全清除羟基自由基。在抑菌活性测试中，研究结果表明，壳聚糖双季铵盐衍生物对黄瓜枯萎致病菌的抑菌率提升了50.0%左右。在1.6 mg/mL 样品浓度下，吡啶环上4位取代产物对芦笋茎枯致病菌和西瓜枯萎致病菌的抑菌率均达到了90.0%。综上所述，壳聚糖及壳聚糖衍生物的抗氧化及抑菌活性按以下顺序排列：壳聚糖双季铵盐衍生物 > 壳聚糖单季铵盐衍生物 > 壳聚糖。随着季铵盐数目的增加，壳聚糖衍生物的抗氧化和抑菌活性均得到有效增强。

虽然壳聚糖双季铵盐衍生物的抗氧化活性和抑菌活性均得到了提高，但是仍有进步的空间。在壳聚糖双季铵盐衍生物的基础上，利用2,3-环氧丙基三甲基氯化铵将2-羟丙基三甲基氯化铵引入到壳聚糖6位羟基位置，进一步增加壳聚糖上季铵盐数目，得到壳聚糖三季铵盐衍生物。在抗氧化活性测试中，壳聚糖三季铵盐抗氧化活性得到进一步增强，在1.6 mg/mL 样品浓度下，对超氧阴离子自由基的清除率提高到95.0%。在抑菌活性测试中，样品浓度为1.6 mg/mL 时，壳聚糖三季铵盐衍生物对芦笋茎枯致病菌、黄瓜枯萎致病菌和西瓜枯萎致病菌三种常见植物致病菌的抑制率均达到了93.0%以上。随着壳聚糖分子中季铵盐数目的进一步增加，得到了具有更高生物活性的产物。

氯乙酰壳聚糖作为一种重要的反应中间体，可以参与三级胺的成盐过程。基于此，将含有不同烷基链长的叔胺及含有不同取代基团的咪唑以盐的形式引入到壳聚糖分子中，在增加壳聚糖季铵盐数目的同时，探究了不同取代基团对壳聚糖生物活性的影响。研究结果表明，与壳聚糖相比，所有含不同链长烷基链的壳聚糖季铵盐衍生物的抗氧化活性和抑菌活性均得到了显著地提高，并且有较短烷基链的产物清除自由基的能力相对较强，因为烷基链越长，供电子能力越强，正电荷密度降低，导致抗氧化活性减小。抑菌活性测试结果表明，在1.6 mg/mL 样品浓度下，所得产物对梨轮纹病菌的抑菌率均大于90.0%。烷基链的脂溶性有助于壳聚糖衍生物与真菌细胞壁中脂溶性物质之间的疏水基-疏水基相互作用。在咪唑类壳聚糖季铵盐的衍生物中，咪唑盐基团的引入增强了壳聚糖衍生物的抗氧化和抑菌活性，特别是在咪唑环上含有氨基和烷基链取代基团的产物。

在本论文中，在壳聚糖季铵盐的基础上进行再修饰，得到了一系列在不同取代位置，含有不同官能团，具有不同季铵盐数目的壳聚糖季铵盐的衍生物。测试结果表明，壳聚糖季铵盐的衍生物抗氧化活性和抑菌活性得到进一步增强，为壳聚糖在农药、医药等领域的开发和利用提供了应用研究的基础。

Chitin is a kind of natural nitrogen-containing polysaccharide with wide source and abundant storage in nature, which has good biocompatibility and biodegradability. Chitosan was obtained by deacetylation of chitin. Chitosan is the only natural alkaline polysaccharide in nature, with non-toxicity, film-forming, hygroscopic moisturizing, antioxidant, antimicrobial and other unique biological activities. But because of the intermolecular and intramolecular hydrogen bonds to enhance the rigid structure of molecules, the solubility of chitosan in water or alkaline solution is very poor. Besides, chitosan itself does not have good antimicrobial and antioxidant activity, which will be unable to meet the needs of efficient development. Chemical modification such as acylation, quaternary ammonium salt, phosphorylation and so on is one of effective means for improving the water solubility and bioactivity of chitosan. Among many chemical modification methods, the quaternized chitosan derivatives are widely concerned because of their good water solubility and higher biological activity.

In this paper, two methods of preparing quaternary ammonium salt chitosan derivatives were used and a series of quaternized chitosan derivatives containing different amounts of quaternary ammonium salts and different active groups were successfully synthesized based on the quaternized chitosan. The structures of the products were characterized by FTIR and NMR spectroscopy, and elemental analysis. The antioxidant activity was tested by removing superoxide radicals, DPPH radicals, and hydroxyl radicals in vitro, and the antifungal activity against several common plant pathogenic fungi including Botrytis cinereal, Phomopsis Asparagi, Gibberella Zeae, Physalospora piricola, Fusarium oxysporum f. sp. cucumerium, and Fusarium oxysporum f. sp. niveum was determined by using in vitro mycelium growth rate test. Chitosan derivatives with higher antioxidant activity or higher antifungal activity were obtained.

Amino polysaccharides often have some special physiological activities such as antioxidant, antimicrobial, anti-tumor, anticoagulant, etc. First, the amino group was alkylated to obtain the N,N,N-trimethyl chitosan derivative (TMC), which could not only increase the positive charge density, but also increase the water solubility of the product in this paper, which is conducive to the further reaction. Therefore, on the basis of TMC, the aminoethyl group was introduced into 6-OH of chitosan by the nitrogen-containing nucleophilic reagent to obtain 6-O/N-(aminoethyl)-2-trimethyl quaternary ammonium salt chitosan derivatives. The antioxidant and antifungal activities were also tested. The antioxidant results showed that the scavenging values of 6-N-(aminoethyl)-2-trimethyl quaternary ammonium salt chitosan derivatives and 6-O-(aminoethyl)-2-trimethyl quaternary ammonium salt chitosan derivatives against superoxide radicals and DPPH radicals increased by about 40.0% at 1.6 mg/mL concentration when compared to chitosan. The scavenging value of 6-N-(aminoethyl)-2-trimethyl quaternary ammonium salt chitosan derivative against hydroxyl radicals was more than 90.0%. However, the antioxidant activity of 6-O/N-(aminoethyl)-2-trimethyl quaternary ammonium salt chitosan derivatives decreased when compared to TMC. The antifungal results showed that the antifungal activity of 6-O/N-(aminoethyl)-2-trimethyl quaternary ammonium salt chitosan derivatives were better than chitosan. For example, the inhibitory value of 6-N-(aminoethyl)-2-trimethyl quaternary ammonium salt chitosan derivative against Botrytis cinereal was 90.0% at 1.0 mg/mL. Besides, the antifungal activity of 6-O/N-(aminoethyl)-2-trimethyl quaternary ammonium salt chitosan derivatives was further enhanced when compared to TMC.

The heterocyclic structure often has special biological activity in the chitosan derivatives. For example, the N heterocyclic pyridine has been applied in pesticides fields because of their good antimicrobial activity . In this paper, the pyridine ring was introduced into the chitosan through the Schiff bases reaction. The N atoms with the tertiary amine property in the pyridine ring could be alkylated. The double quaternary ammonium salts chitosan derivatives containing the pyridine salt were obtained. The results showed that the antioxidant activity of the double quaternary ammonium salts chitosan derivatives was enhanced. The antioxidant ability of the double quaternary ammonium salts chitosan derivatives against DPPH radicals could be comparable to that of Vc at 1.6 mg/mL. And the scavenging ability against hydroxyl radicals was better, even the product with the nitrogen atom on the 3 position of the pyridine ring could completely scavenge hydroxyl radicals. Besides, the inhibitory indices of the double quaternary ammonium salt chitosan derivatives were increased by about 50.0% through the antifungal test. The double quaternary ammonium salts chitosan derivative with the nitrogen atom on the 4 position of the pyridine ring showed 90.0% inhibitory indices against Phomopsis asparagi and Watermelon fusarium at 1.6 mg/mL. In summary, the antioxidant and antifungal activities of chitosan and the chitosan derivatives were listed in the following order: the double quaternary ammonium salts chitosan derivatives > the single quaternary ammonium salt chitosan derivative > chitosan. The antioxidant and antifungal activities of the chitosan derivatives enhanced with the increasing number of quaternary ammonium salt.

Although the antioxidant and antifungal activities of the double quaternary ammonium salts chitosan derivatives have been improved, there is still room for improvement. 2-hydroxypropyl trimethyl ammonium chloride was introduced into 6-OH of chitosan by using 2,3-epoxy propyl trimethyl ammonium chloride to obtain the triple quaternary ammonium salts chitosan derivatives, which will further increase the density of positive charge on the basis of the double quaternary ammonium salts chitosan derivatives. The antioxidant activity reached 95.0% scavenging values at 1.6 mg/mL. The antioxidant test showed that the antioxidant activity of triple quaternary ammonium salt chitosan derivatives was further enhanced. The results showed that the antifungal indices of the triple quaternary ammonium salts chitosan derivatives against Phomopsis asparagi, Fusarium oxysporum, and Watermelon fusarium reached more than 93.0% at 1.6 mg/mL. The bioactivity of the products was higher with increasing the number of the quaternary ammonium salts.

Acetyl chloride chitosan, as an important intermediate, can react with tertiary amine to form salts. The tertiary amine containing different length alkyl chain and the imidazole ring containing different substituted groups were introduced into chitosan molecules in the form of salts. Except for increasing the amount of quaternary ammonium salts, the effects of different substituted groups on the antioxidant and antifungal activities were also investigated. The results showed that the antioxidant and antifungal activities of all the quaternary ammonium salts chitosan derivatives containing different long alkyl chains were significantly higher than that of chitosan. Moreover, the products with shorter alkyl chains showed a relatively stronger scavenging ability, because the longer the alkyl chain is, the stronger the electronic capacity is, the lower the positive charge density would be, which would reduce antioxidant activity. The antifungal results showed that all the products showed more than 90.0% inhibitory indices at 1.6 mg/mL against Physalospora piricola Nose. The lipid solubility of alkyl chain will be helpful to the hydrophobic base-hydrophobic base between chitosan derivatives and fungal cell wall fat-soluble substances. Among the quaternary ammonium salts chitosan derivatives containting imidazole, the introduction of imidazolium enhanced the bioactivity of chitosan derivatives, especially for the products containing amino and alkyl chain substituted groups on the imidazole ring.

In this paper, on the basis of quaternized chitosan, a series of quaternary ammonium salts chitosan derivatives with different replacement positions, different functional groups, and different amounts of quaternary ammonium salts were obtained. The results showed that antioxidant and antifungal activities of quaternary ammonium salts chitosan derivatives were further enhanced, which provides the applied research basis for the further development and utilization of chitosan in pesticide, pharmaceutical and other fields.