【作者】 王新宇；

【导师】 宋慧；

【作者基本信息】 吉林农业大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 小刺猴头[Hericium caput-medusae (Bull.:Fr)Pers.]是一种珍贵的可药用和可食用菌,是长白山自然保护区稀有的菌物资源。小刺猴头菌含有多种营养物质,其中研究较多的是多糖成分,在提高免疫力、抗肿瘤、抗氧化、增强消化功能等方面具有活性。目前,对于小刺猴头菌的研究主要集中在提取方法,结构研究和生物活性上,对小刺猴头菌化学修饰方面的研究甚少。本研究采用两种方法进行硫酸化衍生物的制备,并检测小刺猴头菌多糖及其硫酸化衍生物的生物活性。将推动小刺猴头菌在多糖药物学和生物学方面的发展,为进一步开发小刺猴头菌的药用价值提供理论依据。采用热水浸提法提取小刺猴头液体深层发酵浸膏多糖,在液料比,提取时间和提取温度单因素试验的基础上,利用SAS 9.2(the SAS System for Windows 9.2)软件设计响应面,以多糖得率作为响应值,优化得最佳提取条件：液料比2.29：1(mL/g),提取时间4.52 h,提取温度79.5℃,多糖得率可达12.30%。经乙醇分步沉淀,根据得率选择75%级分进一步纯化,采用Sevag法去除游离蛋白。透析选择MW 8000～10000的透析袋。选用SepharoseCL-6B凝胶柱层析分级分离多糖。多糖纯度鉴定结果显示分离出的多糖为均一组分多糖,命名为HLSF。采用凝胶柱层析法测得多糖的分子量为4.32×104Da。采用氯磺酸-吡啶法(Wolfrom method)和浓硫酸法对HLSF进行硫酸化,通过红外光谱分析,具有硫酸酯键的特征吸收,证明硫酸基团已经与多糖结合,将制得的硫酸化衍生物分别命名为HLSFL和HLSFN。通过体外MTT试验和SOD试剂盒分析检测了硫酸化修饰前后多糖样品的活性,体外MTT试验结果显示经硫酸化修饰后的多糖样品对MCF-7细胞的抑制率均比多糖样品活性高,当多糖样品浓度为1200μg/mL时,氯磺酸-吡啶法硫酸化修饰衍生物对MCF-7细胞的抑制率提高了27.00%,浓硫酸法硫酸化修饰衍生物对MCF-7细胞的抑制率提高了21.21%,结果表明经硫酸化修饰后的多糖样品对MCF-7细胞的抑制率明显增强；SOD试剂盒检测硫酸化修饰前后的多糖样品清除超氧阴离子能力,得出经氯磺酸-吡啶法修饰后的多糖样品比未硫酸化的多糖样品活力提高了102.49%,经浓硫酸法修饰后的多糖样品比未硫酸化的多糖样品活力提高了27.53%,结果表明经硫酸化修饰后多糖样品的抗氧化活性均有所增强,而且经氯磺酸-吡啶法制备的硫酸化衍生物活性要比浓硫酸法制备的硫酸化衍生物活性高一些。综上所述,经硫酸化修饰后的多糖样品生物活性更强,氯磺酸-吡啶法比浓硫酸法更适合制备小刺猴头液体深层发酵浸膏多糖硫酸化衍生物。更多还原

【Abstract】 Hericium caput-medusae is one of the valuable fungi for both medical and edibility, It is scarce fungus natural resources in Mount Changbai natural reserve. Hericium caput-medusae include many kinds of nutritive material, of which polysaccharide component is major studied, its activity has many aspects about increasing immunity、resisting cancer、anti-oxidant、enhancing digestive function and so on. Nowadays, the research about extraction method、structures research、bioactivity aspects, but studying on chemical modification of Hericium caput-medusae on a large scale. This study used two methods to preparation sulfated derivatives, then the biological activities of Hericium caput-medusae and sulfated derivatives were determined. It will impulse the development of Hericium caput-medusae polysaccharide about the aspect of pharmacology and biologic. On development medical value of Hericium caput-medusae to provide accordance with theory.Warm water extraction was used for hericium caput-medusae liquid submerged fermentation extractum polysaccharides, based on solvent-solid ratio、extracting time、extracting temperature single-factor experiments, The SAS 9.2 software was employed to design response surface, the yield of polysaccharides was used as the responsive values, The optimum extraction conditions by optimize were as follow: solvent-solid ratio 2.29:1 (mL/g), extracting time 4.52 h, extracting temperature 79.5℃, the yield of polysaccharides rate was 12.30%. Through ethyl alcohol fractional precipitation, to choose 75% fraction further purification on the base of yield, removing floating protein by Sevag method. Dialysis select bag filter which is MW 8000-10000. Selection Sepharose CL-6B gel column chromatography was used to fractionation on polysaccharides, collecting the main peak, The result of identification the purity of polysaccharide is uniform polysaccharide, named HLSF. Gel chromatography is used to measure the molecular weight of polysaccharide is 4.32×104Da.HLSF was sulfated using Wolfrom method and sulfuric acid method, to obtain HLSFL and HLSFN respectively. The result is analyses by infrared spectrum and substitution, it has the special absorption peak about sulfate, to prove the sulphuric acid group has linked with the polysaccharide, the products has named HLSFL and HLSFN respectively. Through the MTT experiment in vitro and SOD kit analysis comparing the activity of polysaccharide sample which is modified or not, the result of MTT experiment in vitro displayed the polysaccharide sample which is sulfated has more activity to the cell inhibition ratio of MCF-7 than the polysaccharide sample. When the polysaccharide concentration is 1200μg/mL, the inhibition ratio of HLSFL is improving to 27.00% to the cell of MCF-7, the inhibition ratio of HLSFN is improving to 21.21% to the cell of MCF-7, The result of sample polysaccharides inhibition ratio of the activity to the cell of MCF-7 by sulfating modification is enhanced obviously. SOD kit analysis comparing the activity of polysaccharide sample to remove superoxide anion which is modified or not. The activity of HLSFL is improved 102.49% than sample polysaccharide, The activity of HLSFN is improved 27.53% than sample polysaccharide, the result indicated the activity of anti-oxidant of sample polysaccharide by sulfating modification is enhanced obviously. Furthermore the activity of the sulfating derivatives by Wolfrom method is more higher than the activity of the sulfating derivatives by sulfuric acid method. As all the result, the bioactivity of sample polysaccharide sulfate is better, Wolfrom method is more fit to prepare polysaccharide sulfate of Hericium caput-medusae liquid submerged fermentation extractum than the sulfuric acid method.更多还原