平菇多糖的磷酸化修饰及其结构、肝保护作用的研究

【作者】 张扬；

【导师】 朱彩平；

【作者基本信息】 陕西师范大学 ， 食品科学， 2019， 硕士

【摘要】 平菇是常见的食药两用菌,在我国有着悠久的栽培历史,因其鲜美的风味而广受大众喜爱。平菇富含多糖,但平菇多糖(Pleurotus ostreatus polysaccharides,POP)因为其溶解性较差的问题使其在细胞试验中很难发挥其有效作用,诸多报道显示,多糖的结构修饰能够改变多糖的空间构型和生物活性,有关平菇多糖的分子修饰国内外鲜有研究,因此本试验旨在以平菇多糖为研究对象,优化平菇多糖的磷酸化修饰工艺,初步探索磷酸化平菇多糖(Phosphorylated Pleurotus ostreatus polysaccharides,PPOP)的体外抗氧化活性和基本结构特征,深入探究PPOP对四氯化碳诱导的雄性昆明小鼠肝损伤的保护作用,主要结论如下:(1)采用热水浸提法提取POP,以单因素试验对磷酸酯化影响较大的四个因素,进行正交试验设计,得到的最佳磷酸酯化修饰工艺为:三聚磷酸钠:三偏磷酸钠=6:1,反应温度90℃,反应时间3h,溶液pH=9。该方法修饰POP的磷酸化接枝量为9.824%,此法简单易行,磷酸根含量高。(2)POP和PPOP分别经DEAE-52纤维素柱和G-100凝胶柱层析后,POP得到POP-A和POP-B两个组分,PPOP得到PPOP-A、PPOP-B和PPOP-C三个组分。紫外光谱表明磷酸酯化对POP和PPOP的最大吸收波长有影响。刚果红试验表明POP不含多股螺旋结构,而PPOP三维构象发生改变,存在多股螺旋结构。红外光谱试验表明,PPOP经磷酸酯化后保留了多糖的特征吸收峰,检测出了 P=O、P-O-C的吸收峰。环境扫描电镜显示PPOP呈碎片化,形态卷曲,碎片不规则不完整,分子内部作用力减少;分子量试验证明PPOP-A、PPOP-B和PPOP-C的平均分子量分别为17kDa、64kDa和81kDa;HPLC试验表明,PPOP主要是由半乳糖醛酸和木糖组成,其摩尔百分比分别为67.9%和26.1%,PPOP-A由92.3%的半乳糖醛酸和7.6%的木糖构成,PPOP-B主要由41.8%的半乳糖醛酸和57.8%的木糖构成,PPOP-C主要是由6.0%的鼠李糖、6.3%的木糖和86.8%的半乳糖醛酸构成。(3)对上述提取的POP和优化工艺制得的PPOP进行了体外抗氧化试验。DPPH试验显示PPOP在浓度为1mg/mL时,对DPPH自由基的清除率为79.85%,高于同浓度POP的57.35%;羟基自由基清除试验表明在PPOP在浓度为1mg/mL时,PPOP对·OH的清除率达到79.48%,高于同浓度POP的42.09%;超氧阴离子自由基清除试验显示PPOP在浓度为1mg/mL时O2-·清除率为82.52%;ABTS+试验证明当PPOP浓度为1mg/mL时,对ABTS+的清除率达到88.97%,高于同浓度下POP的75.43%;还原力试验证明PPOP的还原能力强于POP。(4)小鼠试验结果表明,PPOP相比POP拥有更强的保肝作用和体内抗氧化活性。不同浓度的PPOP和POP对小鼠进行预防饲喂,肝损伤小鼠体重减轻有所改善,血细胞数量较多,肝脏的肿胀程度缓解,TC、TG、LDL-C浓度降低,HDL-C和ALB浓度增长。血清中ALT、AST和AKP含量降低,肝脏中T-AOC、CAT、T-SOD和GSH-PX活性提高,MDA含量减少。病理学研究显示PPOP组小鼠的肝细胞得到了更好的保护。更多还原

【Abstract】 Pleurotus ostreatus is a common dietetic Chinese herb in China.It has a long history of cultivation in china because its delicious flavor is widely loved by the public.Pleurotus ostreatus is rich in polysaccharides and polysaccharides is also one of the main active ingredients of Pleurotus ostreatus.However,Pleurotus ostreatus polysaccharides(POP)cannot exert its effective effect in the cell tests because of its solubility problem.The structural modification of polysaccharides can change the spatial configuration and biological activity.The molecular modification of POP has rarely been reported at home and abroad.So this experiment aims to optimize the phosphorylation modification process by using Pleurotus ostreatus polysaccharides as research object and explore the in vitro antioxidant activity and basic structural features preliminarily.Then we studied the protective effect of PPOP on liver injury induced by carbon tetrachloride in male Kunming mice deeply.The main conclusions are as follows:(1)The POP were extracted by hot water.Then we researched four facts which have greater impact on phosphorylation by single factor experiments and deal the orthogonal test.The best phosphate modification process is:sodium tripolyphosphate:sodium trimetaphosphate=6:1,reaction temperature 90℃,reaction time 3 h,solution pH=9.The phosphorylation grafting amount modified by this method is 9.824%.The method is simple and easy,and the phosphate content is high.(2)After separated by DEAE-52 and G-100,POP was divided into POP-A and POP-B and PPOP was divided into PPOP-A,PPOP-B and PPOP-C.The UV spectrum indicates that phosphating has an effect on the maximum aborption wavelength of UV.The Congo red test showed that POP does not contain a multi-strand structure while PPOP has multi-strand structure.FT-IR showed that PPOP retained the characteristic absorption peak of polysaccharides after phosphate esterification,and the absorption peaks of P=O and P-O-C were detected.The ESEM showed that PPOP was fragmented,the shape was curled,the fragments were irregular and the internal forces were reduced.The molecular weight test proved that the average molecular weight of PPOP-A,PPOP-B and PPOP-C was 17kDa、64kDa and 81 kDa.HPLC experiments showed that PPOP was mainly composed of 67.9%galacturonic acid and 26.1%xylose.PPOP-A consists of 92.3%galacturonic acid and 7.6%xylose.PPOP-B consists of 41.8%galacturonic acid and 57.8%xylose.PPOP-C consists of 6.0%rhamnose,6.3%xylose acid and 86.8%galacturonic.(3)An in vitro antioxidant test was carried out on the POP and PPOP.The DPPH test showed that the scavenging rate of DPPH free radicals was 79.85%when PPOP was 1 mg/mL,which was higher than 57.35%of the same concentration POP.The ·OH radical test showed that when PPOP was at a concentration of 1 mg/mL,the scavenging rate of ·OH reached 79.48%,which was higher than 42.09%of POP at the same concentration.The O2-·test showed that the O2-·scavenging rate of PPOP was 82.52%at a concentration of 1 mg/mL.The ABTS+ test proved that when the PPOP concentration was 1 mg/mL,the clearance rate for ABTS+ reached 88.97%,which was higher than 75.43%of POP at the same concentration.The reducing power test verified that the reduction ability of PPOP is stronger than POP.(4)The results of mouse experiments showed that PPOP has stronger hepatoprotective effect and antioxidant activity in vivo than POP.Different concentrations of PPOP and POP were used to pretreat mice.The body weight of the mice was improved.The number of blood cells increased.The degree of swelling of the liver was relieved.The concentrations of TC,TG and LDL-C were decreased,and the concentrations of HDL-C and ALB were increased.The levels of ALT,AST,and AKP in mice serum were decreased.T-AOC,CAT,T-SOD,and GSH-PX activities of the liver were increased,and the content of MDA decreased.Pathological studies showed that PPOP protect hepatocytes better than POP.更多还原