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墨鱼黑色素的酶提优化、结构解析及降血糖和抗病毒活性研究
宋雯
Subtype博士
Thesis Advisor邢荣娥
2024-05-19
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学博士
Degree Discipline海洋生物学
Keyword真黑素 酶提取技术 结构解析 II 型糖尿病 杜兰病毒
Abstract

黑色素是一种多功能天然生物聚合物,广泛分布于动物、植物和微生物中,因其结构独特,天然、无毒副作用,具有降血糖、抗病毒、抑菌、抗肿瘤等多种生物学活性,在药物研发应用方面备受关注。但因黑色素结构复杂,仅溶解在碱性溶液中,且传统的提取方法以碱溶解和酸沉淀为主,严重破坏了黑色素的结构,限制了黑色素的应用。II型糖尿病是一种慢性代谢性疾病,患者的胰岛素功能受损,导致血糖水平升高。如果不进行有效控制,糖尿病会引发多种严重并发症,如心血管疾病、肾脏疾病、神经系统病变等,给患者的健康和生活质量造成严重影响。II型糖尿病的发病率呈现不断增加的趋势,特别是随着全球人口老龄化和生活方式变化,糖尿病在全世界已经成为一个严重的公共卫生问题。现有治疗药物包括促胰岛素分泌药物、胰岛素类药物、胰岛素增敏药物、糖苷酶抑制剂等,易产生低血糖和胃肠道反应等副作用,远远不能满足临床需要,因此开发安全、有效控制糖尿病的药物迫在眉睫。流行病学研究表明,II型糖尿病增加了更频繁、更严重的病毒感染风险。抗病毒I型促炎细胞因子会助长胰岛素抵抗,成为II型糖尿病发展的危险因素,这表明在II型糖尿病背景下,免疫和内分泌系统之间存在着诸多相互影响的互动。药物的吸收及肠道健康状态也是影响糖尿病症状的重要因素。肠道免疫系统是身体的重要组成部分,它在维护免疫平衡、抵御病原体等方面发挥着重要作用。人类诺如病毒(Human Norovirus,hNoV)是全球急性胃肠炎最常见的原因之一,具有很强的传播性与扩散性。由于目前无法在实验室中培养足够病毒滴度的hNoV,而且缺乏有效的动物培养模型。为了研究hNoV,人们研究开发了可普遍使用且可培养的具有相同感染机制的替代病毒,如杜兰病毒(Tulane Virus,TV)。因此,本文以墨鱼汁为原料,优化黑色素的提取技术,探索黑色素的结构,并研究黑色素的降血糖及抗病毒活性及机制,为墨鱼下脚料墨鱼汁的高值利用以及黑色素作为糖尿病药物的开发提供理论基础。具体研究结果如下:

建立了复合酶从墨鱼汁中提取黑色素的工艺。通过单酶、双酶和三酶提取筛选,经过单因素及响应面优化分析,发现以碱性蛋白酶、中性蛋白酶、风味蛋白酶的三酶提取方法(Alkaline-Neutral-Flavored Protease, ANF)可以获得68 %提取率且纯度达92 %的黑色素(ANF)。采用红外光谱、紫外扫描光谱、电子自旋共振、拉曼光谱、X射线衍射、扫描电子显微镜、原子力显微镜与冷冻电镜技术对黑色素的结构进行鉴定,表明酶法提取的黑色素为真黑素,是由颗粒大小为 100-200 nm的稳定球型纳米颗粒聚集而成。本研究继续深入探究了墨鱼汁中黑色素的结构,揭示了其组成和组装过程,发现黑色素先形成基础30 nm的纳米颗粒球体,这些球体进一步组装成长链结构,最终长链盘旋聚集并进一步包裹形成直径约100-200 nm的球型纳米颗粒,且内部具有一定的空间压力。这一发现可深化对真黑素结构动力学的理解,为未来研究其性质和应用奠定了基础。

对黑色素物理化学性质及稳定性进行了分析,结果表明,ANF在碱性溶液中溶解,中性溶液中微溶,酸性溶液中基本不溶,常见有机试剂中不溶,与原料墨鱼汁粉末相比,ANF在碱溶液中的溶解度提高。且ANF在不同的光照、紫外线辐射、温度和食品添加剂条件下都能保持稳定,适用于在化妆品、食品、药品方面的开发。

在酶、细胞和动物水平上,研究了ANF的降血糖活性。酶抑制活性表明,ANF可显著抑制α葡萄糖苷酶活性,优于阿卡波糖,对具有V型凹陷结合位点的α-淀粉酶抑制活性一般;在人肝癌细胞系(HepG2)胰岛素抵抗模型中,ANF增强了葡萄糖利用和肝糖原合成;在 II型糖尿病ICR小鼠模型中对ANF的降血糖活性进一步评估表明,在0.04 g·kg-1的剂量下,ANF在缓解高血糖、降低糖化血清蛋白水平、改善糖耐量、调节总胆固醇和低密度脂蛋白水平以及抗氧化指数方面具有显著作用,其降糖活性优于常用降糖药物拜糖平和二甲双胍。而且,ANF对肾细胞的密度和浸润状态有积极影响,同时减轻肝细胞的炎症扩大和变形,并可维持胰腺细胞的生存,而不会对小鼠产生任何不良反应。此外,在对于ANF改善糖尿病合并抑郁症的研究中,ANF对糖尿病小鼠的挣扎能量水平提高和减少静止时间表现出积极影响,对小鼠海马体神经元紧密性具有显著改善。

高血糖可导致氧化应激,通过诱导β细胞功能障碍和胰岛素抵抗促进II型糖尿病的发病。本文在自由基清除、细胞水平以及体内肝组织抗氧化指标上研究了ANF的抗氧化活性,并与四聚体结构预测相关联。与黑色素标准品(Melanin Standard, MelS)相比,ANF具有更好的自由基清除活性,可清除89.62 %的DPPH自由基、96.79 %的超氧阴离子自由基、93.20 %的羟自由基,能更好地修复细胞氧化损伤。0.04 g·kg-1的ANF可显著增强小鼠体内的抗氧化酶(SOD、GSH-Px)、降低丙二醛水平。基于分子能量、分子静电势和高斯计算的分子轨道,预测真黑素中含有具有抗氧化活性的四聚体结构为DHICA3-DHI1'''和“DHICA3-DHI1”。

进一步研究了0.04 g·kg-1的ANF对糖尿病小鼠尿液代谢及肠道菌群的影响。结果显示,糖尿病小鼠组与正常组相比,在尿液代谢组成上存在显著差异,ANF处理的糖尿病小鼠组表现出明显的代谢调节效果,尿液中部分代谢物,如氨基酸及其衍生物、脱落酸及其衍生物、α-酮酸及其衍生物等得到了调节。表明黑色素可能通过影响这些代谢物的水平来调节糖尿病小鼠的代谢状态。将代谢差异物基于KEGG通路富集分析,发现黑色素可影响多个代谢通路,如氨基酸代谢、葡萄糖稳态、溶酶体通路等,表明黑色素可能通过调节代谢通路来发挥其治疗糖尿病的作用。采用16S rRNA基因测序分析了ANF对糖尿病小鼠肠道菌群的影响。Alpha多样性分析中稀疏曲线分析表明样本覆盖了微生物群落的大部分成分,Beta多样性分析中黑色素组与糖尿病组之间的微生物群落结构存在差异,并且经过ANF给药后显示出明显的改善。粪便菌群物种组成发现,黑色素组中Firmicutes相关丰度显著增加,Bacteroidetes相关丰度显著降低,Proteobacteria丰度也有所下降,这表明ANF可能通过改变菌群结构来治疗糖尿病小鼠。在菌属水平的热图分析中,发现了一些菌属在糖尿病组中富集,而ANF则显著下调这些菌属。表明了ANF可通过调节肠道菌群的功能从而改善糖尿病症状,为其开发成为糖尿病药物进一步提供了理论依据。

此外,本文研究了ANF对诺如病毒替代品杜兰病毒(Tulane Virus,TV)的抗病毒活性。研究表明,ANF在低于1000 μg/mL时没有细胞毒性,且在0.977 μg/mL时具有显著的抗病毒作用,其最佳饱和作用浓度为 62.5 μg/mL。进一步详细研究了ANF与细胞的相互作用,从表面相互作用到细胞内途径,揭示了它们的分层组装和潜在的受控药物释放机制。ANF孵育后的转录组分析揭示了基因表达和抗病毒信号通路激活的显著变化,为细胞防御随后的病毒入侵做好准备,而不会产生不良影响。ANF激活抗病毒机制,表现出明显更高IFN-α 和 IFN-β的表达。特别地,ANF在抗TV活性研究中,转录组学分析结果发现ANF于3 h即可激活糖尿病信号通路。由于ANF其具有优越的降血糖活性,因此研究推测其在不同细胞类型中可能通过激活糖尿病的信号通路以及相关抗病毒信号通路发挥作用。综上所述,ANF抗病毒活性与其降血糖活性之间存在着密切的相互作用关系,这为进一步研究其在糖尿病治疗和抗病毒药物开发方面提供了重要线索。

Other Abstract

Melanin is a multifunctional natural biopolymer widely distributed in animals, plants, and microorganisms. Due to its unique structure, natural, non-toxic nature, it has attracted much attention in the field of drug development due to its various biological activities such as hypoglycemic, antiviral, antibacterial, and anticancer effects. However, due to the complex structure of melanin, it only dissolves in alkaline solutions, and traditional extraction methods mainly rely on alkaline dissolution and acid precipitation, which severely damage the structure of melanin, limiting its application. Type II diabetes is a chronic metabolic disease characterized by impaired insulin function, leading to elevated blood sugar levels. If not effectively controlled, diabetes can lead to various serious complications such as cardiovascular diseases, kidney diseases, neurological disorders, etc., severely affecting the health and quality of life of patients. The incidence of type II diabetes is continuously increasing, especially with the global aging population and lifestyle changes, making diabetes a serious public health issue worldwide. Current treatment drugs include insulin secretagogues, insulin mimetics, insulin sensitizers, α-glucosidase inhibitors, etc., which are prone to side effects such as hypoglycemia and gastrointestinal reactions, far from meeting clinical needs. Therefore, the development of safe and effective drugs to control diabetes is urgently needed.

Epidemiological studies have shown that type II diabetes increases the risk of more frequent and severe viral infections. Antiviral type I proinflammatory cytokines contribute to insulin resistance, becoming a risk factor for the development of type II diabetes, indicating that there are many interactions between the immune and endocrine systems in the background of type II diabetes. The absorption of drugs and the health status of the intestines are also important factors affecting the symptoms of diabetes. The intestinal immune system is an important part of the body, playing a crucial role in maintaining immune balance and resisting pathogens. Human Norovirus (hNoV) is one of the most common causes of acute gastroenteritis worldwide, with strong infectivity and spread. Since it is currently impossible to cultivate enough virus titers of hNoV in the laboratory, and there is a lack of effective animal models for cultivation. In order to study hNoV, researchers have developed universally usable and cultivable alternative viruses with the same infection mechanism, such as Tulane virus (TV). Therefore, this study used squid ink as a raw material to optimize the extraction technology of melanin, explore the structure of melanin, and study the hypoglycemic and antiviral activities and mechanisms of melanin, providing a theoretical basis for the high-value utilization of squid ink waste and the development of melanin as a drug for diabetes. The specific research results are as follows:

Established a process for extracting melanin from squid ink with composite enzymes. Through single-factor and response surface optimization analysis, it was found that the three-enzyme extraction method of alkaline protease, neutral protease, and flavor protease (ANF) could obtain a melanin extraction rate of 68% with a purity of 92% (ANF). The structure of melanin was identified by infrared spectroscopy, ultraviolet scanning spectroscopy, electron spin resonance, Raman spectroscopy, X-ray diffraction, scanning electron microscopy, atomic force microscopy, and cryo-electron microscopy techniques, indicating that the melanin extracted by enzyme method is true melanin, composed of stable spherical nanoparticles with a particle size of 100-200 nm. This study further explored the structure of melanin in squid ink, revealing its composition and assembly process, and found that melanin first formed basic 30 nm nanospheres, which further assembled into long-chain structures, and finally, long-chain spirals aggregated and further enveloped to form spherical nanoparticles with a diameter of about 100-200 nm, with a certain internal pressure. This discovery deepens the understanding of the structural dynamics of true melanin and lays the foundation for future research on its properties and applications.

The physical and chemical properties and stability of melanin were analyzed, and the results showed that ANF dissolved in alkaline solution, slightly soluble in neutral solution, basically insoluble in acidic solution, and insoluble in common organic reagents. Compared with raw squid ink powder, ANF had increased solubility in alkaline solution. Moreover, ANF could maintain stability under different light, ultraviolet radiation, temperature, and food additive conditions, suitable for development in cosmetics, food, and pharmaceuticals.

At the enzyme, cellular, and animal levels, the hypoglycemic activity of ANF was studied. Enzyme inhibition activity showed that ANF could significantly inhibit α-glucosidase activity, superior to acarbose, and generally inhibited α-amylase activity with a V-shaped pocket. In the insulin-resistant model of human hepatoma cells (HepG2), ANF enhanced glucose utilization and hepatic glycogen synthesis. Further evaluation of the hypoglycemic activity of ANF in ICR mice with type II diabetes model showed that at a dose of 0.04 g·kg-1, ANF had significant effects in alleviating hyperglycemia, reducing glycated serum protein levels, improving glucose tolerance, regulating total cholesterol and low-density lipoprotein levels, and antioxidant index. Its hypoglycemic activity was superior to commonly used hypoglycemic drugs pioglitazone and metformin. Moreover, ANF had a positive effect on the density and infiltration status of renal cells, relieved the enlargement and deformation of hepatic cells, and could maintain the survival of pancreatic cells without causing any adverse reactions in mice. In addition, in the study of ANF improving depression in diabetic mice, ANF showed positive effects on the struggling energy level increase and reduced immobility time in diabetic mice, and significantly improved the tightness of hippocampal neurons in mice.

High blood sugar can lead to oxidative stress, promoting the onset of type II diabetes by inducing β-cell dysfunction and insulin resistance. This study investigated the antioxidant activity of ANF at the level of free radical scavenging, cellular level, and in vivo liver tissue antioxidant indicators, and correlated with tetramer structure prediction. Compared with melanin standard (MelS), ANF had better free radical scavenging activity, could scavenge 89.62% of DPPH radicals, 96.79% of superoxide anion radicals, and 93.20% of hydroxyl radicals, and could better repair cell oxidative damage. 0.04 g·kg-1 ANF significantly enhanced the antioxidant enzymes (SOD, GSH-Px) in mice, and reduced the level of malondialdehyde. Based on molecular energy, molecular electrostatic potential, and Gaussian calculation of molecular orbitals, it was predicted that true melanin contained tetramer structures with antioxidant activity DHICA3-DHI1''' and "DHICA3-DHI1".

Furthermore, the effect of 0.04 g·kg-1 ANF on urine metabolism and intestinal flora of diabetic mice was studied. The results showed that there were significant differences in urine metabolic composition between the diabetic mouse group and the normal group, and the ANF-treated diabetic mouse group showed significant metabolic regulation effects, regulating some metabolites in urine, such as amino acids and their derivatives, ketones and their derivatives, etc. This indicates that melanin may regulate the metabolic status of diabetic mice by affecting the levels of these metabolites. Based on KEGG pathway enrichment analysis of differentially metabolized substances, it was found that melanin could affect multiple metabolic pathways, such as amino acid metabolism, glucose homeostasis, lysosome pathways, etc., indicating that melanin may exert its therapeutic effect on diabetes by regulating metabolic pathways. The effect of ANF on the intestinal flora of diabetic mice was analyzed by 16S rRNA gene sequencing. Sparse curve analysis in alpha diversity analysis showed that the samples covered most of the components of the microbial community, and beta diversity analysis showed differences in the microbial community structure between the melanin group and the diabetes group, with significant improvement after ANF administration. The composition of fecal microbial species found that the abundance of Firmicutes was significantly increased, while the abundance of Bacteroidetes and Proteobacteria was significantly decreased in the melanin group, indicating that ANF may treat diabetic mice by changing the structure of the gut microbiota. In the heatmap analysis at the genus level, some genera were found to be enriched in the diabetic group, while ANF significantly down-regulated these genera. This indicates that ANF may improve the symptoms of diabetes by regulating the function of the intestinal flora, providing a theoretical basis for its further development as a drug for diabetes.

In addition, this study investigated the antiviral activity of ANF against the surrogate virus Tulane virus. The study showed that ANF had no cytotoxicity at concentrations below 1000 μg/mL and had significant antiviral activity at 0.977 μg/mL, with the optimal saturation concentration being 62.5 μg/mL. Further detailed studies on the interaction between ANF and cells, from surface interactions to intracellular pathways, revealed their layered assembly and potential controlled drug release mechanisms. Transcriptome analysis of ANF-incubated cells revealed significant changes in gene expression and activation of antiviral signaling pathways, preparing cells for defense against subsequent viral invasion without adverse effects. ANF activated the antiviral mechanism and exhibits significantly higher expression of IFN-α and IFN-β. In the study of ANF antiviral activity against TV, transcriptomic analysis results showed that ANF could activate the diabetes signaling pathway as early as 3 hours. Since ANF has superior hypoglycemic activity, it is speculated that it may exert its effects through the activation of the diabetes signaling pathway and related antiviral signaling pathways in different cell types. In summary, there is a close interaction between the antiviral activity of ANF and its hypoglycemic activity, providing important clues for further research on its treatment of diabetes and the development of antiviral drugs.

MOST Discipline Catalogue理学
Pages180
Language中文
Table of Contents

目 录

第1章 绪论 1

1.1 黑色素国内外研究进展 1

1.1.1 黑色素种类 1

1.1.2 黑色素的提取方法 4

1.1.3 黑色素的结构解析和理化性质 6

1.1.4 黑色素的生物学活性 8

1.2 墨鱼汁黑色素研究概况 12

1.2.1 墨鱼汁资源概述 14

1.2.2 墨鱼汁的营养与药用价值 15

1.2.3 墨鱼汁黑色素资源的利用现状及存在的问题 15

1.3 糖尿病概述 16

1.3.1 糖尿病分类 18

1.3.2 糖尿病并发症 18

1.3.3 糖尿病的治疗现状 19

1.4 病毒性胃肠炎影响糖尿病治疗 22

1.4.1 病毒性胃肠炎 23

1.4.2人诺如病毒的研究现状 23

1.4.3人诺如病毒替代物——杜兰病毒 24

1.5 研究背景与意义 25

1.6 研究内容与技术路线 25

1.6.1 研究内容 25

1.6.2 技术路线 26

第2章 墨鱼汁中黑色素的酶复合提取及结构、理化性质解析 27

第一节 酶提取黑色素工艺优化 27

1 实验材料与仪器 27

1.1 实验材料 27

1.2 仪器设备 28

2 实验方法 28

2.1 最佳酶种类筛选 28

2.2 单酶提取工艺优化 28

2.3 双酶提取工艺 29

2.4 三酶提取工艺 29

2.5 统计方法 29

3 结果与讨论 30

3.1 提取黑色素酶筛选结果 30

3.2 单酶提取黑色素的单因素优化及响应面优化研究 31

3.3 黑色素的双酶提取和三酶提取结果 39

4 小结 39

第二节 黑色素结构鉴定及四聚体模拟优化 41

1 实验材料与仪器 42

1.1 实验材料 42

1.2 仪器设备 42

2 实验方法 43

2.1 傅里叶变换红外光谱 43

2.2 紫外-可见光全波长扫描 43

2.3 扫描电子显微镜 43

2.4 电子自旋共振 43

2.5 固态核磁共振 43

2.6 激光拉曼光谱 44

2.7 X射线衍射光谱 44

2.8 原子力显微镜 44

2.9 冷冻透射电子显微镜 44

2.10 动态光散射 45

2.11 真黑素单体键合合成四聚体的结构优化 45

2.12 H2O2氧化荧光法测定纯度 45

2.13 溶解度检测 45

2.14 元素组成分析 45

2.15 稳定性分析 45

2.16 统计分析 46

3 结果与讨论 46

3.1 红外光谱分析 46

3.2 紫外全波长扫描 48

3.3 扫描电子显微镜 49

3.4 电子自旋共振光谱 51

3.5 固态核磁共振光谱 52

3.6 激光拉曼光谱 53

3.7 X射线衍射光谱 53

3.8 原子力显微镜 54

3.9 冷冻透射电子显微镜 55

3.10 动态光散射 56

3.11 真黑素单体键合合成四聚体的结构优化 58

3.12 H2O2氧化荧光法测定纯度 60

3.13 溶解度检测 61

3.14 元素组成分析 62

3.15 稳定性分析 62

4 小结 64

第3章 黑色素对糖尿病及合并并发症治疗研究 65

1实验材料与仪器 65

1.1 实验材料 65

1.2 仪器设备 66

2 实验方法 67

2.1 黑色素对关键酶的抑制作用 67

2.2 黑色素对HepG2细胞胰岛素抵抗模型的改善 67

2.3 黑色素对II型糖尿病小鼠体内降血糖活性研究 68

2.4 黑色素对II型糖尿病小鼠体内外抗氧化活性研究 70

2.5 黑色素对 II型糖尿病小鼠体内抗糖尿病合并抑郁症活性研究 72

2.6 黑色素处理的糖尿病小鼠尿液代谢组学、肠道微生物菌群组成分析 73

2.7 动物伦理 73

2.8 统计方法 73

3 结果与讨论 73

3.1 细胞毒性 73

3.2 黑色素对关键酶的抑制作用 74

3.3 黑色素对HepG2细胞IR模型的改善 75

3.4黑色素对II型糖尿病小鼠降血糖活性研究 77

3.5 黑色素对II型糖尿病小鼠体内外抗氧化活性研究 84

3.6 黑色素对 II型糖尿病小鼠体内抗糖尿病合并抑郁症活性研究 87

3.7 黑色素处理的糖尿病小鼠尿液代谢组学分析及信号通路富集 91

3.8 黑色素处理的糖尿病小鼠肠道菌群16S rRNA基因测序结果 97

4 小结 103

第4章 黑色素抗人诺如病毒替代物——杜兰病毒活性研究 105

1实验材料与仪器 105

1.1 实验材料 106

1.2 仪器设备 106

2 实验方法 106

2.1 细胞毒性 106

2.2 TCID50方法筛选抗病毒活性机制 107

2.3光学显微镜观察CPE状态及细胞对黑色素的摄取 107

2.4 TV的逆转录-定量聚合酶链反应(RT-qPCR)分析 108

2.5 扫描电子显微镜 108

2.6 原子力显微镜 108

2.7 透射电子显微镜 109

2.8 激光共聚焦活细胞成像 109

2.9 RNA-seq差异基因表达 109

2.10 一氧化氮(NO)定量及IFN-α/β的RNA表达 110

2.11统计方法 110

3 结果与讨论 111

3.1 细胞毒性 111

3.2 TCID50方法筛选抗病毒活性机制 112

3.3 细胞对黑色素的摄取及光学显微镜观察 113

3.4 扫描电子显微镜观察纳米颗粒与细胞表面作用 115

3.5 原子力显微镜 118

3.6 透射电子显微镜 118

3.7 利用TIRF进行实时SR旋转盘共焦显微镜观察 120

3.8 RNA-seq差异基因表达 121

3.9 GO通路注释及富集分析 124

3.10 KEGG通路注释及富集分析 126

3.11 抗病毒信号通路 127

4 小结 129

第5章 结论与创新点 131

本文结论 131

创新点 132

参考文献 133

致谢 153

作者简历及攻读学位期间发表的学术论文与其他相关学术成果 155

Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/185230
Collection实验海洋生物学重点实验室
中国科学院海洋研究所
Recommended Citation
GB/T 7714
宋雯. 墨鱼黑色素的酶提优化、结构解析及降血糖和抗病毒活性研究[D]. 中国科学院海洋研究所. 中国科学院大学,2024.
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墨鱼黑色素的酶提优化、结构解析及降血糖和(8542KB)学位论文 暂不开放CC BY-NC-SA
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