壳聚糖硫酸酯化产物的合成及其抗凝血性能研究

【作者】 梁媛媛；

【导师】 胡星琪；

【作者基本信息】 西南石油学院 ， 应用化学， 2005， 硕士

【摘要】 甲壳素是自然界生物合成量仅次于纤维素的天然高分子化合物,每年生物合成量约100亿吨,壳聚糖是甲壳素的脱乙酰基产物。二者作为广泛存在的多聚糖资源,具有无毒、可生物降解等优异性质,如何充分利用这一宝贵的资源一直是人们探索的重要课题。在众多的衍生物当中,甲壳素、壳聚糖硫酸化修饰成为最为诱人的领域,由于甲壳素、壳聚糖具有与肝素相似的骨架结构,比其他多糖更适合制备类肝素物质。肝素系硫酸化的糖胺聚糖,是临床应用重要的抗凝药物之一,不仅能够有效地阻止血液凝固,防止血栓的形成,还能澄清血浆脂质,降低血胆固醇含量,其主要来源于哺乳动物如牛、羊等的肝、心、脾等脏器;存在价格昂贵,提取困难等缺点,并且由于其具有较高的抗凝活性,在临床应用上存在用量控制困难等问题,常引起过度溶血和血小板含量减少等副作用。因此我们考虑将壳聚糖硫酸酯化改性,希望获得与肝素结构相似,分子参数可调节因此其抗凝血功能也可能调节的新型类肝素药物,从而有望取代肝素在临床上得到应用。 壳聚糖分子中C₂位上的—NH₂及C₃、C₆位上的—OH均具有较强的反应活性,在酯化试剂的作用下,均可发生反应。本文拟将C₆-OH作为壳聚糖酯化反应的活性点,并选择适当的硫酸酯化试剂,以生成物S含量为考察指标,通过正交试验确定最佳试验条件。同时考察壳聚糖原料的两个重要的分子参数——脱乙酰度和分子量对壳聚糖硫酸酯S含量以及抗凝活性的影响,通过IR确定产物的结构。最后测定壳聚糖硫酸酯的TT（凝血酶原时间）,对其抗凝血活性进行表征和分析。研究取得以下进展: （1）设计了分段洗涤法,采用氢氧化钠与壳聚糖进行多次脱乙酰化反应,制备了一系列高脱乙酰度（DD>90%）且分子量变化微小的壳聚糖精样。 （2）提出中性条件滴加H₂O₂短时间反应的方法,通过反应温度控制产物分子量。制备的壳聚糖精样脱乙酰几乎相同,但分子量随反应温度而降低（M_η=328,700～64,000）。 （3）通过正交试验得到硫酸/氯磺酸混酸体系高硫含量产物的条件:酯化试剂为硫酸/氯磺酸=1:2（VN）,反应温度35℃,反应时间8h,反应体系中壳聚糖浓度为28.57g/L,产物S含量为11.57%。 （4）红外光谱证明本试验条件下壳聚糖硫酸酯化反应活性位为C₆-O,通过与肝素的红外特征谱带波数比较证明了本试验合成的壳聚糖硫酸酯化产物与肝素分子结构相似。 （5）初步探索了硫酸/氯磺酸混酸酯化壳聚糖的反应机理。 （6）通过TT的测定,证明壳聚糖酯化产物具有抗凝活性,C₆-O是抗凝活性点,最高抗凝活性为24.3U/mg,其抗凝作用是通过与ATⅢ结合形成复合物,该复合物灭活FⅡa、Ⅹa来实现。 （7）揭示壳聚糖硫酸酯分子参数与其抗凝血功能的关系:揭示类肝素物质——壳聚糖硫酸酯抗凝活性不仅与S含量有关,还与其分子结构有密切关系:a)其他分子参更多还原

【Abstract】 Chitosan is a biopolymer and consists of β（1→4）-2-amino-2-deoxy-D-glucose repeat units. It is normally obtained by the alkaline deacetylation of chitin, which is the second most abundant, naturally occurring structural material found in the shells of crustacean and cuticles of incects and also in the cell walls of some fungi and microorganisms. The sulfation of chitosan is the most interesting field of its chemical modification, since chitosan has the similar structure with heparin, which is of high antithrombin activity and is popularily used in medical field. Moreover, instead of heparin, the using of chitosan haparinoids can reduce the risk of contamination. Therefore, our work was carried out to investigate and determinate the optimum reactive condition of sulfated chitosan. The structure of sulfated chitosan was charactered by IR spectra. From its antithrombin activities, we studied its antithrombin mechanism and reached a qualitative conclusion. The main achievements our work had obtained were as follows:1. The chitosan with high DD （degree of deacetylation） ranging from 83.45% to 99.76% can be obtained by the method of interval washing with NaOH and the molecular weight of chitosan was scarcely affected.2. The method of H₂O₂ degradation didn’t influence the degree of deacetylation of chitosan; the molecular weight of degraded chitosan was ranging from 328,700 to 64,000.3. The optimum synthenic condition of sulfated chitosan reaction of chitosan was about 8 hours, 35℃ and H₂SO₄:HSO₃Cl （V:V） was 1:2, this sulfated chitosan had the highest S-content,4. The result of IR spectrum showed that the sulfated chitosan is 6-O-sulfated chitosan and its main structure had something in common with the heparin.5. Explore elementarily the mechanism of the reaction between H₂SO₄/HSO₃Cl and chitosan.6. The 6-0 sulfated chitosan showed antithrombin activities in vitry by TT （thrombin time） investigation and the best antithrombin activity was 24.3U/mg, though it was less than equivalent heparin and the does of the heparinoid could be easily controlled. Moreover, we can deduce from the fact of lengthened-TT that the mechanism of antithrombin activity of chitosan was to combine with AT—HI to restrain the activities of FKa, IXa and II a, therefore , the TT can be able to be lengthened.7. The relationship between structrare of the 6-O sulfated chitosan and the antithrombin activities had been investigated as follows: a) The antithrombin activity tendency of the sulfated chitosans with same DD showed as 110.5KD>32.3KD>78.7KD b) the DD of the sulfated chitosans had obvious influences on the antithrombin activities when the molecular weight of the samples did’t varied. The tendency was 93.45%>83.45%>77.65%更多还原