【作者】 杨曦明；

【导师】 张丽萍；

【作者基本信息】 东北师范大学 ， 生物化学与分子生物学， 2011， 博士

【摘要】 白树花[Polyporus albicans （Imaz.） Teng]主要分布于中国吉林省,属于担子菌纲多孔菌科多孔菌属,是一种可食用和药用的真菌。目前对白树花多糖的研究报道较少,只有国内对其结构和免疫活性进行了初步研究。但是白树花多糖的精确结构研究以及对多糖进行硫酸化修饰后的抗凝血活性研究未见报道。因此本研究将白树花真菌多糖进一步分离纯化,对其结构、硫酸化修饰及修饰后多糖的抗凝血活性进行研究,以期为白树花多糖的进一步开发利用提供理论基础。白树花粗多糖经冻融、醇分级、蛋白酶法和Sevage法脱蛋白、HPLC分离后得到一个主要的纯化组份-PATPⅠ。经HPLC分析表明:白树花多糖PATPⅠ由葡萄糖、甘露糖、半乳糖组成,其摩尔比为15.7:4.2:1,分子量为33 kDa。采用部分酸水解、高碘酸氧化、Smith降解、甲基化、GC-MS、IR、NMR等方法对PATPⅠ进行测定分析,确定其分子结构为:主链由（1→2）-β-D-Man残基、以及少量的（1→3）α-Glc构成,分支点为（1→2, 6）-β-Glc残基;支链丰富,平均每2个己糖残基就会出现一个分支。推断PATPⅠ结构的重复单元为:采用氯磺酸-吡啶法对白树花多糖进行了硫酸化修饰,结果显示:多糖硫酸化修饰后红外光谱（IR）出现1250cm^-1的S=O特征吸收峰;Sepharose CL-6B柱层析结果表明硫酸化修饰后多糖样品的洗脱峰峰位前移,即硫酸基的引入使多糖的分子量明显增大,多糖中的部分羟基被硫酸酯基团所取代。离子色谱法分析测得硫酸基占14.7%,取代度为Ds=1.41。体外抗凝血研究结果表明,白树花多糖硫酸酯具有良好的抗凝血效果;其抗凝血功能主要通过抑制内源凝血系统的凝血因子发挥作用,与肝素的抗凝血机理基本一致,即通过与抗凝血酶Ⅲ（ATⅢ）结合,进一步抑制凝血因子Ⅱa和Ⅹa而发挥抗凝血作用;动物体内抗凝血实验结果与体外实验结果是一致的,而且在使用12小时内未发现对动物血液、肝肾功能有不良影响。肝素在防止血栓形成及治疗方面在临床上广泛使用,但是存在较多的副作用,因此寻找肝素的替代品一直是备受关注的研究课题。本研究结果显示白树花多糖硫酸酯抗凝血效果及初步的安全评价均比较良好,具有替代肝素成为新的抗凝药的潜力,值得进一步深入研究。更多还原

【Abstract】 Polyporus albicans (Imaz.) Teng is a traditional Chinese medicine and edible fungus distributed in Jilin Province of China, which is a Polyporaceae fungus belonging to the Basidiomycetes. There are a few studies of the structure and immunobiological activity of Polyporus albicans (Imaz.) Teng have been reported, but its anti-coagulation activities after sulfate modification are still unknown. Our study is on Polyporus albicans (Imaz.) Teng purification, structural analysis and the anti-coagulation activity after sulfate modification. These findings indicate a huge potential clinical use of Polyporus albicans (Imaz.) Teng.PTPI, one of the most proportional Polyporus albicans(Imaz.) Teng polysaccharide, was obtained by graded freezing, alcohol- precipitation, enzymatic and Sevage deproteinization and HPLC purification. The HPLC data indicated that PTPI was composed of glucose, galactose and seminose residues in the ratio of 15.7:4.2:1, with molecular weight of 3.3×10~4 Da,.PTPI was analyzed by acid hydrolysis, periodate oxidation, Smith degradation, methylation, GC-MS, IR, NMR to identify the structure. The results showed that the main chain of PTPI is composed of (1→2)-β-D-Man residues and a few (1→3)α-Glc, Man residues. PTPI is branched at the (1→2, 6)-β-Glc every two hexose residues along the main chain. The following structure indicates the repeated units of PTPI:Next, we modified the Polyporus albicans (Imaz.) Teng polyose by the method of chlorosulfonic acid-pyridine. The IR spectra of sulfate modified polysaccharides present a S=O characteristic absorption at 1250 cm–1. The data of the Sepharose CL-6B column chromatography showed a unique and high molecular weight shifted elution peak, which caused by the addition of sulfate into polysaccharide. It suggested that partial hydroxyl groups of polysaccharide were substituted with sulfate. The content of sulfur in polysaccharide was 14.7%, and degree of substitution of sulfate was 1.41.The anti-coagulant assay of Polyporus albicans (Imaz.) Teng sulfate (PATS) indicates its remarkable anticoagulant activity in vitro. The study on anticoagulation mechanism suggests that PATS got involved in the intrinsic pathway. The anti-coagulation activity of PATS was due to the inhibition of the coagulation factors IIa and Xa activities ediated by antithrombin III(ATIII). The anti-coagulation mechanism of PATS is absolutely identical to that of heparin. In vivo assay suggests that there is no adverse effect on animal blood, liver and kidney after taking PATS for 12 hrs.Heparin is the most popular anticoagulant used in clinic, however, its side effects have also caused highly concern. It is still under intensive investigation to synthesize effective heparin substitute. In this study, PATS has the similar anti-coagulation characteristic to heparin, but with a better anti-coagulation effect. PATS has more bio-safety advantage because of deriving from edible fungus-polysaccharide. Therefore, PATS has promising future to be developed and used as an ideal substitute for heparin in clinic.更多还原