中国科学院海洋研究所机构知识库

海洋是生命的摇篮，大量的生物蕴含其中。海洋环境与陆地环境存在着较 大的差异，这使得海洋动物进化出独特的代谢机制，海洋动物的多糖结构往往 较为新颖，有别于陆生动物，展现出更加丰富的结构和生物活性。海洋底栖动 物多糖作为一种独特的生物活性物质，因其生物活性和医疗应用前景，已引起 科学家的广泛关注。这些特有的生物学活性可能来自其独特的分子结构。单环 刺螠和方格星虫均为海洋底栖动物，富含多糖、脂肪酸和氨基酸，具备很高的 营养价值；据报道，单环刺螠多糖具有抗氧化、降血糖和预防糖尿病等功效； 而方格星虫多糖具有抑菌、抗缺氧、抗辐射和免疫调节等活性，两者都有着很 高的药用价值。然而，目前对两种海洋底栖动物多糖，尤其是单环刺螠多糖的 结构及构效关系仍然大部分未知。据此，本文以单环刺螠和方格星虫为原料， 采取了一系列的提取和分离纯化手段，从单环刺螠及方格星虫体壁中分别得到 了数种新颖的多糖组分；在对这些多糖的理化性质和结构进行了测定后，对其 抗凝血活性展开了研究，主要研究结果如下： 1.对单环刺螠体壁组织分别采取了 100℃和 60℃热水浸提，依次在 30%、 50%和 80%的乙醇浓度下进行醇沉，得到了蛋白含量 5%以下的 6 种粗多糖组分。 后经离子色谱纯化后，共得到 11 种不同的多糖，包括 5 种葡聚糖（UHG-1, UHG-2, UHG-3, ULG-1, ULG-2），6 种糖胺聚糖（UHA-1, UHA-2, UHA-3, ULA1, ULA-2, ULA-3）。 在单糖组成的检测中，UHG-1、2 和 ULG-1、2 中的葡萄糖含量达 97%以上， 剩余的约 3%则由少量的半乳糖、半乳糖醛酸和葡糖胺构成。相较之下，UHG-3 中的葡萄糖虽然仍占据主导（73.90%），但其他成分如葡糖胺、半乳糖、果糖和 甘露糖含量也较为可观，分别占比 8.57%、6.25%、3.74%和 3.35%。此外， UHA-1、2和ULA-1、2中岩藻糖的含量较高，分别为27.37%、33.65%、18.88% 和 26.61%，这一发现为这些多糖的特定功能和应用提供了线索。 分子量测定结果显示，在 100℃提取条件下，中性多糖（UHG）和糖胺聚 糖（UHA）的分子量表现出随乙醇浓度上升逐渐降低的趋势。而在 60℃提取条 件下，葡聚糖 ULG-1、2 的分子量也遵循相似的降低规律。整体上，60℃提取 条件下多糖的分子量都相应的大于 100℃提取的多糖。这些结果明确表明，提 取温度及乙醇浓度对单环刺螠多糖的分子量具有显著影响。 为了探明多糖的聚集状态，我们利用扫描电子显微镜（SEM）对单环刺螠 多糖的表面结构进行了观察。结果显示，高分子量的葡聚糖（UHG-1 和 ULG-1） 呈现出类似球形的自组装结构；而 UHG-2、UHG-3 和 ULG-2 则展现出清晰的片状结构。所有 UHA 和 ULA 组分（UHA-1、2、3；ULA-1、2、3）均表现为片 状结构，其超分子自组织形态可能与其生物活性有关。 单环刺螠的葡聚糖核磁共振结果显示，其很具有类似糖原及淀粉的多分支 结构。除了由 α-1,4 键以及 α-1,6 键之外，单环刺螠体壁很可能含有 β 构象的葡 聚糖。在 FTIR 图谱中，可以观察到 930 cm-1附近出现了一个肩峰，这个肩峰的 位置对应 β-1,3 糖苷键。同时根据碘结合实验结果，单环刺螠体壁的大分子量葡 聚糖是不同于淀粉以及糖原的葡聚糖，且其一级结构中应不存在较长 α-1,4 片段， 结合前期的结构数据，我们推测该葡聚糖可能具有较糖原更频繁的分支结构， 且有能含有 β-葡聚糖或 β-葡聚糖片段。 单环刺螠糖胺聚糖的核磁结果显示，其中含有多种类型的糖苷键，包括 α糖苷键（δ 5.33, 5.10, 5.01）和 β-糖苷键（δ 4.88, 4.79, 4.57, 4.55）。同时碳谱中 δ 69.37和 δ 69.85处的两个明显的信号提示单环刺螠体壁糖胺聚糖样品可能含有硫 酸基团取代。FTIR 的结果显示，1242 cm-1 附近的峰对应 S=O 的伸缩振动，804 cm-1 附近的峰对应 C–O–S 的伸缩振动。同时，13C NMR 谱图中这两个信号可能 分别对应于两种不同环境下的硫酸基团，说明硫酸基团的取代位置并不单一。 抗凝血活性探究中，UHA-3 表现出较好的 APTT 活性，剩余多糖无明显的 APTT、PT 和 TT 活性。根据相对分子量结果，UHA-3 的 Mn是 11 种多糖中最小 的，仅为 4200 Da，我们推测较小的分子量更有利于其发挥 APTT 活性。同时单 环刺螠多糖的抗凝血活性可能与海参多糖和海星多糖类似，其岩藻糖基被一定 量的硫酸根所修饰，在抗凝血活性中发挥着重要的作用。 2. 采用分级醇沉、复合酶解和 DEAE-52 离子交换层析从方格星虫体壁中分 离纯化了一种非中性多糖，并采用 HPLC、GPC、SEM、FTIR 等技术对其结构 特征进行表征，同时评价了其体外抗凝血活性。结果表明，在优化提取条件下， 方格星虫多糖(SLP)的产率为 5.76%，总糖含量为 90.24%。HPLC 分析表明 SLP 主要由 Man、GlcN、Gal 和 Glc 组成，摩尔比为 1: 2.83: 5.34: 6.95。GPC 测得 SLP 的 Mn 为 9201 g/mol，PDI 为 1.05，表明其为均一多糖。SEM 和 FTIR 分析 显示 SLP呈不规则片状结构，其主要以 α-吡喃糖构象存在。SLP表现出对 APTT 的延长作用，在 200–400 µg/mL 时呈现量效关系，但 800 µg/mL 时活性趋于饱 和。目前，SLP 的抗凝活性尚不及海参等海洋硫酸化多糖，推测与其硫酸基含 量低有关，这也暗示了 SLP 可能具有不同于其他海洋多糖的抗凝血机制，值得 进一步研究。 本论文的研究成果，对单环刺螠多糖和方格星虫多糖的结构研究提供了理 论基础，同时对其抗凝血活性展开的表征有助于进一步开发其作为抗血栓药物 的潜在价值，对海洋底栖动物资源的开发和利用具有一定的借鉴意义。

The ocean is the cradle of life, harboring an immense amount of biodiversity. Significant differences exist between the marine environment and terrestrial environments, leading to the evolution of unique metabolic mechanisms in marine animals. The polysaccharide structures of marine animals are often novel and distinct from those of terrestrial animals, exhibiting richer structural and biological activities. As a unique bioactive substance, marine benthic animal polysaccharides have attracted widespread attention from scientists due to their biological activities and medical application prospects. These specific biological activities may stem from their unique molecular structures. Both the Urechis unicinctu and the Sipunculus nudus L. are marine benthic animals rich in polysaccharides, fatty acids, and amino acids, making them highly nutritious. It is reported that polysaccharides from the Urechis unicinctu possess antioxidant, hypoglycemic, and diabetes-preventive effects, while those from the Sipunculus nudus L. exhibit antibacterial, anti-hypoxia, anti-radiation, and immunomodulatory activities. Both species possess high medicinal value. However, much remains unknown about the structures and structure-activity relationships of polysaccharides from these two marine benthic animals, particularly those from the Urechis unicinctu. Therefore, this study aimed to extract and purify novel polysaccharide components from the body walls of both the Urechis unicinctu and the Sipunculus nudus L. using a series of extraction and purification techniques. After determining the physicochemical properties and structures of these polysaccharides, their anticoagulant activities were investigated. The main research findings are as follows: 1. The polysaccharides of the Urechis unicinctus body wall were extracted using hot water at temperatures of 100°C and 60°C. Simultaneously, alcohol precipitation was carried out at 30%, 50%, and 80% ethanol concentrations, resulting in six crude polysaccharide fractions. After ion exchange chromatography purification, a total of 11 different polysaccharides were obtained, including 5 glucans and 6 glycosaminoglycans. Glucose was the predominant monosaccharide in UHG-1, 2, ULG-1, 2, constituting over 97%, indicating their glucan structural characteristics. The remaining approximately 3% consisted of small amounts of galactose, galactose aldonic acid, and glucosamine. In contrast, glucose in UHG-3 accounted for 73.90%, with other components such as glucosamine, galactose, fructose, and mannose also present. Notably, there were significant ion interactions between the anionic components UHA1, 2, 3, and ULA-1, 2, 3 and the DEAE resin. UHA-1, 2, and ULA-1, 2 exhibited high levels of fucose, accounting for 27.37%, 33.65%, 18.88%, and 26.61%, respectively, providing clues for specific functions and applications of these polysaccharides. Molecular weight analysis revealed a decreasing trend in neutral polysaccharides (UHG)and anionic polysaccharides (UHA) under 100°C extraction conditions. Similarly, the molecular weights of ULG-1, 2 decreased under 60°C extraction conditions. The molecular weight of ULA-2 was prominent among the glycosaminoglycans, followed by ULA-1 and ULA-3. These results indicated a significant impact of extraction temperature on the molecular weight and structural characteristics of Urechis unicinctus polysaccharides. Scanning electron microscopy (SEM) was used to observe the surface structure of the polysaccharides, revealing spherical aggregates for high molecular weight glucans (UHG-1 and ULG-1) and distinct sheet-like structures for UHG-2, UHG-3, and ULG-2. All UHA and ULA components displayed sheet-like structures, providing a basis for further understanding their biological activities and potential applications. Preliminary structural studies using infrared spectroscopy and nuclear magnetic resonance analysis were conducted on these 11 polysaccharides. The results showed that all polysaccharide samples contained monosaccharides with hydroxyl and carbonyl functional groups, possibly including carboxyl and aldehyde structures. NMR results indicated the presence of a branched structure similar to glycogen and starch in the glucan of Urechis unicinctus. Besides the glucan composed of α-1,4 and α-1,6 bonds, the body wall of Urechis unicinctus likely contains β-configuration glucan. In the FTIR spectrum, a shoulder peak appeared near 930 cm-1 , corresponding to the β1,3 glycosidic bond. According to the results of iodine-binding experiments, the highmolecular-weight glucan in the body wall of Urechis unicinctus differs from starch and glycogen, and its primary structure should not contain long α-1,4 fragments. Combining previous structural data, we speculate that this glucan may have a more frequent branching structure than glycogen and may contain β-glucan or β-glucan fragments. NMR analysis of the glycosaminoglycan from Urechis unicinctus revealed the presence of multiple types of glycosidic bonds, including α-glycosidic bonds (δ 5.33, 5.10, 5.01) and β-glycosidic bonds (δ 4.88, 4.79, 4.57, 4.55). Additionally, two distinct signals at δ 69.37 and δ 69.85 indicated the possible presence of sulfate groups in the glycosaminoglycan samples from the body wall of Urechis unicinctus. This conclusion was further supported by FTIR results, specifically, the peak near 1242 cm-1 corresponded to the stretching vibration of S=O, and the peak near 804 cm-1 corresponded to the stretching vibration of C–O–S. Furthermore, the 13C NMR spectrum suggested that these two signals might correspond to sulfate groups in two different environments, indicating that the substitution positions of sulfate groups are not uniform. During the investigation of anticoagulant activity, UHA-3 exhibited significant APTT activity, while the remaining polysaccharides did not display notable APTT, PT, or TT activity. Based on the results of relative molecular weight measurements, UHA3 had the smallest Mn among the 11 polysaccharides, with a value of only 4200 Da. We speculate that a smaller molecular weight may contribute to its APTT activity. Additionally, the anticoagulant activity of polysaccharides from the Urechis unicinctusmay be similar to those from sea cucumber and starfish. The fucosyl groups in these polysaccharides, which are modified by a certain amount of sulfate groups, play a crucial role in anticoagulant activity. 2. A non-neutral polysaccharide was isolated and purified from the body wall of Sipunculus nudus using graded alcohol precipitation, compound enzymatic hydrolysis, and DEAE-52 ion exchange chromatography. The structural characteristics of this polysaccharide were characterized using HPLC, GPC, SEM, and FTIR techniques, and its anticoagulant activity in vitro was evaluated. The results showed that under optimized extraction conditions, the yield of Sipunculus nudus polysaccharide (SLP) was 5.76%, and the total sugar content was 90.24%. HPLC analysis revealed that SLP was mainly composed of Man, Rib, Gal, and Glc, with a molar ratio of 1: 2.83: 5.34: 6.95. The Mn of SLP measured by GPC was 9201 g/mol, with a PDI of 1.05, indicating its homogeneity. SEM and FTIR analysis showed that SLP exhibited an irregular lamellar structure and existed mainly in the α-pyranose conformation. SLP exhibited a prolonging effect on APTT, showing a dose-effect relationship at concentrations of 200- 400 µg/mL, but the activity tended to saturate at 800 µg/mL. Currently, the anticoagulant activity of SLP is inferior to that of marine sulfated polysaccharides such as sea cucumber, presumably due to its low sulfate content. This suggests that SLP may have a different anticoagulant mechanism from other marine polysaccharides, deserving further investigation.

第1章 绪论  1

1.1 海洋底栖动物多糖的活性研究背景    1

1.1.1 抗氧化、抗衰老活性  1

1.1.2 抗肿瘤活性  1

1.1.3 抗凝血活性  2

1.1.4 免疫调节活性     2

1.1.5 抗病毒活性  3

1.1.6 调节肠道微生物活性  3

1.1.7 肝脏保护活性     3

1.2 多糖研究概括 4

1.2.1 多糖的提取  4

1.2.1.1 水提法      4

1.2.1.2 酸提法      5

1.2.1.3 碱提法      5

1.2.1.4 酶解提取法      5

1.2.1.5 超声辅助提取法      6

1.2.2 多糖的结构分析  7

1.2.2.1 单糖组成分析   7

1.2.2.2 相对分子量测定      7

1.2.2.3 扫描电镜分析   8

1.2.2.4 红外光谱分析   8

1.2.2.5 核磁共振波谱分析   9

1.3 单环刺螠的研究背景    9

1.3.1 概述     9

1.3.2 形态特征     10

1.3.3 生存环境     11

1.3.4 硫化物耐受机制  12

1.3.5 生物活性物质     12

1.4 方格星虫的研究背景    13

1.4.1 概述     13

1.4.2 形态特征     13

1.4.3 营养价值     14

1.4.4 方格星虫活性多糖     14

1.5 本课题的立题背景和研究思路    15

第2章 单环刺螠体壁多糖的分离提取、结构分析和活性测定     18

2.1 材料与仪器    18

2.1.1 材料     18

2.1.2 仪器     19

2.2 实验方法 20

2.2.1 单环刺螠体壁多糖的提取  20

2.2.2 粗多糖的化学组成测定     20

2.2.3 单环刺螠体壁多糖的分离纯化  21

2.2.4 单糖组成分析     21

2.2.5 相对分子量测定  22

2.2.6 扫描电子显微镜观测  23

2.2.7 傅里叶变换红外光谱  23

2.2.8 核磁共振波谱分析     23

2.2.9 碘结合实验  23

2.2.10 抗凝血活性测定       23

2.2.11 数据分析   24

2.3 实验结果与讨论    24

2.3.1 粗多糖理化性质测定  24

2.3.2 单环刺螠体壁多糖的分离纯化  26

2.3.3 单糖组成分析     27

2.3.4 相对分子量测定  33

2.3.5 扫描电子显微镜观测  39

2.3.6 傅里叶变换红外光谱  41

2.3.7 核磁共振波谱分析     43

2.3.8 碘结合实验  44

2.3.9 抗凝血活性测定  46

2.4 本章小结 46

第3章 方格星虫体壁多糖的分离提取、结构分析和活性测定     48

3.1 材料与仪器    48

3.1.1 材料     48

3.1.2 仪器     48

3.2 实验方法 48

3.2.1 方格星虫多糖的提取  48

3.2.2 理化性质测定     48

3.2.3 方格星虫体壁多糖的分离纯化  48

3.2.4 单糖组成分析     48

3.2.5 相对分子量测定  48

3.2.6 扫描电子显微镜观测  48

3.2.7 傅里叶变换红外光谱  48

3.2.8 抗凝血活性测定  48

3.2.9 数据分析     48

3.3 结果与讨论    49

3.3.1 粗多糖理化性质测定  49

3.3.2 方格星虫体壁多糖的分离纯化  49

3.3.3 单糖组成分析     50

3.3.4 相对分子量测定  51

3.3.5 扫描电子显微镜观测  52

3.3.6 傅里叶变换红外光谱  52

3.3.7 抗凝血活性测定  53

3.4 本章小结 54

第4章 总结与展望     56

4.1  总结      56

4.2  展望      57

参考文献       58

致  谢    68

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    69