中国科学院海洋研究所机构知识库

    褐藻硫酸多糖组分因其具有抗氧化、抗肿瘤、抗炎、抗凝血等多种生物活性，受到了广泛的关注。其中的褐藻多糖硫酸酯组分研究较多， 但对硫酸杂多糖组分关注较少。 本实验室在前期实验中通过对数十种海藻多糖神经保护作用筛选，得到一种对帕金森病（Parkinson’s Disease, PD）具有显著作用的海带低硫酸化杂多糖（Sulfated Hetero-Polysaccharides, UF）。 UF 是一种硫酸根含量较低、单糖组成复杂的杂多糖。 由于其组成复杂， 质量控制困难， 药物代谢动力学进展缓慢， 已成为制约 UF 药物开发过程中的瓶颈。
    采用高效液相色谱法初步建立了海带低硫酸化杂多糖的指纹图谱质量控制技术，对 UF 水解后的单糖组成色谱图进行分析，共鉴定出甘露糖、甘露糖醛酸、鼠李糖、岩藻糖等 8 个主要共有特征峰，且全谱相似度评价进一步证实了该方法
应用于 UF 样品质量控制的可行性。
    针对低硫酸化杂多糖的结构特征，以异硫氰基荧光素（FITC） 为标签，制备得到荧光标记多糖 FITC-UF。通过紫外-可见光谱、红外光谱等分析方法，对制备得到的 FITC-UF 样品进行分析， 表明 FITC 已经成功连接到了 UF 样品上， 荧光强度较强且稳定。 CCK-8 法测定细胞活力实验表明荧光标记样品 FITC-UF 对PC12 细胞无细胞毒性，可用于后续动物实验。方法学实验表明该方法可准确、快速、灵敏的对海带低硫酸化杂多糖样品进行微量、 定量检测，为后续药物代谢动力学实验中大量生物样本的检测奠定了基础。
    经腹腔注射单次给药后，在小鼠的血液及各脏器中均检测到了海带低硫酸化
杂多糖。药物代谢动力学数据分析可得，药物在体内吸收较快，达峰非常迅速；
在体内吸收程度较好，但代谢较快。 海带低硫酸化杂多糖在各器官中的分布比例
较大，且能透过血脑屏障，在脑组织中有一定的蓄积。
    海带低硫酸化杂多糖质量控制方法的建立与初步的药物代谢动力学研究为解决制约 UF 成药过程中的瓶颈问题提供了一定的理论基础，可以在一定程度上加快 UF 作为创新型药物的研发进程。

    Sulfated polysaccharides extracted from brown algae have been paid much attention due to their diverse biological activities such as anti-oxidation, anti-tumor, anti-inflammatory and anticoagulaiton. There are many studies focus on fucoidan, but the sulfated hetero-polysaccharides are less concerned. In our early study of dozens of brown algae polysaccharides, a novel sulfated hetero-polysaccharides (UF) was shown to have significant neuroprotective effects. UF was measured to be a heteropolysaccharide with complex components and low sulfate content. Their quality control and pharmacokinetics is relatively difficult because of its complex composition and has become a cottleneck factor restricting drug development of UF.
    The fingerprint of UF was preliminarily established by HPLC. 8 peaks were identified as D-mannose, D-Mannuronic acid, L-rhamnose, D-glucuronic acid, Dglucose, D-galactose, D-xylose and L-fucose. The similarity evaluation for chromatographic fingerprint indicated the method can be applied to the quality controlof UF.
    Concerning the structural characteristics of UF, a fluorescent labelled polysaccharide FITC-UF was prepared by using isothiocyanate fluorescein (FITC) as label. The analysis results of UV –Vis and IR spectroscopy proved that FITC had been
successfully connected to UF. The fluorescence intensity of FITC-UF was detected to be strong and stable. CCK-8 cytotoxicity test showed that FITC-UF sample had no cytotoxicity on PC12 cells and could be used in animal experiments subsequently. The results of methodology validation proved method to be accurate, rapid and sensitive and can be a foundation for detecting a large amout of biological samples in pharmacokinetic experiments.
    UF sample was detected in serum and organs of ICR mices after intraperitoneal administration. The pharmacokinetic datas showed UF was absorbed rapidly in the body and had obvious concentration accumulation in a short period of time. The concentration of UF entering body circulation was high but its metabolism rate was
high too.The proportion of UF in organs was large. The detection of UF in brain illustrated that UF could be transported through blood-brain barrier and had a certain accumulation in brain.
    The establishment of the quality control method and the results of preliminary pharmacokinetics study for sulfated hetero-polysaccharide extracted from Saccharina japonica can provide a theoretical basis for solving the important problems in the
process of UF drug development. These results also can speed up the development of UF as an innovative drug to a certain extent.
 

目 录
摘 要......................................................................................................... I
ABSTRACT............................................................................................... II
第一章 绪论............................................................................................ 1
1 研究背景..............................................................................................................1
2 中药及其制剂中多糖的质量控制方法..............................................................1
2.1 总糖含量检测方法........................................................................................1
2.2 分子量大小及其分布测定............................................................................3
2.3 单糖组成及含量测定....................................................................................3
3 多糖的神经保护活性机制..................................................................................6
3.1 抗氧化活性与 Nrf2/HO-1 信号通路............................................................6
3.2 MAPK 信号通路..........................................................................................7
3.3 促进神经细胞突起生长................................................................................7
3.4 神经营养作用：提高神经细胞的存活率....................................................7
3.5 NF-κB 家族 ...................................................................................................8
3.6 PI3K/Akt 信号通路.......................................................................................8
3.7 其他神经营养通路的激活............................................................................8
4 多糖的药代动力学研究方法..............................................................................9
4.1 色谱法............................................................................................................9
4.2 荧光标记法....................................................................................................9
4.3 放射性同位素标记法..................................................................................10
4.4 生物测定法..................................................................................................11
5 选题意义与研究内容........................................................................................11
第二章 海带低硫酸化杂多糖 (UF) 水解单糖高效液相色谱指纹图
谱研究....................................................................................................... 13
1 材料和仪器........................................................................................................13
1.1 实验材料......................................................................................................13
1.2 实验仪器......................................................................................................13
2 实验方法............................................................................................................14
2.1 样品制备......................................................................................................14
2.2 理化性质分析..............................................................................................14
2.3 指纹图谱分析..............................................................................................15
2.4 数据分析......................................................................................................17
3 结果与讨论........................................................................................................17
3.1 理化性质分析..............................................................................................17
3.2 水解及衍生化条件优化..............................................................................17
3.3 方法学考察..................................................................................................20
3.4 指纹图谱的建立与分析..............................................................................27
4 结论与展望........................................................................................................32
第三章 海带低硫酸化杂多糖 (UF) 的微量分析方法研究 .............. 33
1 材料和仪器........................................................................................................33
1.1 实验材料......................................................................................................33
1.2 实验仪器......................................................................................................33
2 实验方法............................................................................................................34
2.1 荧光标记 UF 样品的制备 ..........................................................................34
2.2 FITC-UF 中 FITC 取代度的测定..............................................................34
2.3 FITC-UF 的光谱学特性 ............................................................................35
2.4 FITC-UF 细胞毒性试验 ............................................................................35
2.5 方法学考察..................................................................................................36
2.6 数据分析......................................................................................................36
3 结果与讨论........................................................................................................37
3.1 FITC-UF 中 FITC 取代度的测定..............................................................37
3.2 FITC-UF 的光谱学特性 ............................................................................38
3.3 FITC-UF 细胞毒性实验 ............................................................................40
3.4 方法学考察..................................................................................................41
4 结论与展望........................................................................................................45
第四章 海带低硫酸化杂多糖 (UF) 的药代动力学初步研究 .......... 46
1 材料与仪器........................................................................................................46
1.1 实验材料......................................................................................................46
1.2 实验仪器......................................................................................................46
2 实验方法............................................................................................................47
2.1 小鼠药代动力学实验方法..........................................................................47