杏鲍菇多糖的分离纯化及其抗氧化和抗肿瘤的功能研究

【作者】 王宁；

【导师】 杨兴斌；

【作者基本信息】 陕西师范大学 ， 粮食、油脂及植物蛋白工程， 2015， 硕士

【摘要】 目前,食用菌多糖的研究已经成为国内外的热点,研究表明多糖是食用菌中的最有价值活性成分之一,具有抗肿瘤、抗病毒、降血糖和增强免疫力等生物活性。杏鲍菇(Pleurotus eryngii)学名刺芹侧耳,为担子菌类侧耳科(Pleurotaceae)侧耳属,属于食用菇菌类。本文着重考察了杏鲍菇多糖纯化后的体外抗氧化、抗肿瘤活性,阐明了杏鲍菇多糖清除自由基以及抗肿瘤细胞的机制,为杏鲍菇多糖的开发应用提供了理论基础及技术支持。研究采用水提醇沉法来提取杏鲍菇中的粗多糖,提取率为5.4%,多糖总糖含量为61.3%。再经过对杏鲍菇粗多糖进行Sevag除蛋白、活性炭吸附脱色处理,最终得到无色脱蛋白杏鲍菇粗多糖。通过HPLC-UV检测技术来分析杏鲍菇粗多糖(PEP)的单糖组成,结果显示,PEP由8种单糖组成：甘露糖(9.8%)、核糖(1.6%)、鼠李糖(0.15%)、葡萄糖醛酸(0.8%)、葡萄糖(62.8%)、木糖(0.05%)、半乳糖(24.4%)、海藻糖(0.4%),其中葡萄糖、半乳糖以及甘露糖含量较多。由此说明,PEP是由8个单糖组分组成的杂多糖。通过一系列自由基的抗氧化实验,系统评价了的杏鲍菇多糖PEP的体外抗氧化能力,表明PEP具有一定的体外抗氧化活性,并且呈现出较好的剂量依赖效应。对杏鲍菇粗多糖进行DEAE-52纤维素柱层析,并采用G-100葡聚糖凝胶层析柱对得到的多糖组分进行进一步的纯化,DEAE-52纤维素柱层析洗脱后得到2种多糖组分且均为均一的组分,分别命名为PEP-1、PEP-2。紫外扫描结果显示,260-280 nm之间并没有吸收峰,表明PEP-1、PEP-2中不含核酸和蛋白质。红外光谱结果表明,在PEP-1、PEP-2的结构中均存在吡喃糖环结构。通过HPLC-UV对PEP-1、PEP-2的单糖组成进行进一步的检测,结果显示,组成PEP-1的单糖有甘露糖(mannose)、葡萄糖(glucose)以及半乳糖(galactose),组成PEP-2的单糖同样为甘露糖(mannose)、葡萄糖(glucose)以及半乳糖(galactose)。经过高效液相凝胶渗透色谱分析,得到PEP-1、PEP-2的分子量分别为25362D,463396 Da。通过MTT、LDH实验可以得知,与PEP-1相比,PEP-2对人肝癌细胞HepG2有明显的抑制增长作用,且与浓度呈量效关系。DAPI染色发现,这些凋亡使细胞出现了染色质凝集,核碎裂和调亡小体出现等特征。Annexin V-FITC/PI检测PEP-2引起的凋亡结果显示,经PEP-2处理的细胞在6 h之后,就出现了20.49%的早期凋亡,晚期凋亡占到4.27%；而在24 h后,早期凋亡达到35.71%,晚期凋亡也增加到了20.63%,由流式细胞仪检测得出,PEP-2将人肝癌细胞HepG2的生长阻滞在了S期。利用流式细胞仪检测PEP-2诱导的HepG2细胞的活性氧水平,结果显示,随着作用时间的增长,细胞内H2O2水平即得到了明显的升高,因此初步推断,在PEP-2发挥抗肿瘤作用的时候,ROS起到了介导细胞凋亡的作用。本实验从杏鲍菇中提取分离纯化得到2种多糖组分,并对其结构进行了初步的检测与观察,并筛选得到一种组分PEP-2(分子量为46 WDa)具有显著的抑制肝癌细胞的增殖作用,其中ROS在过程中起到了介导凋亡的作用。这一研究发现对研究多糖的抗肿瘤的机制指明了思考的方向,并为功能性食品因子或药物的开发提供了理论依据。更多还原

【Abstract】 Researches of mushroom polysaccharide have become a hot spot in China and abroad, they have shown that polysaccharides is one of the most valuable of the active ingredient in the edible and medicinal mushroom, they have many biological activities, such as anti-tumor, anti-cancer, lowering blood glucose and strengthening the immune system and other. Pleurotus (Pleurotus eryngii), is from Basidiomycetes Pleurotaceae (Pleurotaceae) Pleurotus, edible mushrooms belong to the class.This paper examined the anti-oxidation, anti-tumor activities of the polysaccharide of purified Pleurotus eryngii, clarified the Pleurotus eryngii polysaccharide radical scavenging and anti-tumor effect, supported for the development and application, provided a theoretical foundation and technical of Pleurotus eryngii polysaccharide.PEP was extracted from the P. eryngii used a multistep purification procedure including hot-water extraction and repeated ethanol precipitation. The extraction rate of the dried PEP reached approximately 5.4%(w/w). The total carbohydrate content of PEP was determined to be 61.3%. The compositional monosaccharides of PEP were identified by HPLC-UV technique. The results showed that PEP was composed of D-mannose, D-ribose, L-rhamnose, D-glucuronic acid, D-glucose, D-xylose, D-galactose and D-fucose with the corresponding mole percentage was 9.8%,1.6%,0.15%,0.8%, 62.8%,0.05%,24.4% and 0.4%, respectively. Which D-glucose, D-galactose and D-mannose content more. It was shown that PEP is a heteropolysaccharide composed of eight monosaccharide composition. Through a series of anti-oxidation experiments, the antioxidant capacity was evaluated in vitro, indicated that PEP has antioxidant activities in vitro, and exhibited dose-dependent effects.DEAE-52 cellulose column chromatography and Sephadex G-100 chromatography column used to purified the Pleurotus eryngii polysaccharides for further study. DEAE-52 cellulose column chromatography eluted by two kinds of polysaccharides which were homogeneous components, named PEP-1, PEP-2. UV scanning results showed that,260-280 nm had no absorption peak, and the PEP-1, PEP-2 does not contain nucleic acid and protein. The infrared spectra showed that pyranose ring structure were present in the PEP-1 and PEP-2. Through the monosaccharide composition of HPLC-UV for further testing, the results showed that, monosaccharide composition of PEP-1 contain mannose, glucose and galactose, monosaccharide composition of PEP-2 contain mannose, glucose and galactose. Analysis by HPLC gel permeation chromatography, the molecular weight of PEP-1 is 25362 Da, PEP-2 is 463396 Da, respectively.Through the MTT and LDH assay, PEP-2 had more obvious effect of growth inhibition on human hepatocellular carcinoma HepG2 cells compared with PEP-1, and the activities showed dose effect relationship with the concentration. DAPI staining showed that HepG2 cells appeared apoptosis, and the chromatin condensation, nuclear fragmentation and apoptotic body characteristics. The apoptosis assay used Annexin V-FITC/PI, PEP-2 treated cells in 6 h, then appeared 20.49% of early apoptosis, late apoptosis accounted for 4.27%; and after 24 h, early apoptosis reached 35.71%, late apoptosis also increases to 20.63%. Obtained by flow cytometry, PEP-2 arrested in the human hepatocellular carcinoma HepG2 cells in S phase.The used of flow cytometry in ROS levels of PEP-2-induced HepG2 cells showed that as time goes on, intracellular H2O2 levels were significantly increased, therefore we concluded that, when the PEP-2 exert an anti-tumor effect, ROS-mediated the HepG2 cells apoptosis.In this study, two kinds of polysaccharides separated and purificated from Pleurotus eryngii, and their structures was preliminary testing and observation, and screened to obtain a component inhibiting PEP-2 (molecular weight is 35 W Da) with significant proliferation of liver cancer cells. ROS plays a mediating role in cell apoptosis. The result of our study pointed out the direction thinking of the study of antitumor mechanism of polysaccharides in the future, and provided a theoretical basis for the development of functional food factors or drugs.更多还原