绣球菌多糖的分离纯化及其生物活性研究

发布时间：2019-01-23 18:43

【摘要】：水提取法提取绣球菌粗多糖,进行脱色和脱蛋白处理,分离纯化出多糖组分,测其单糖组成与红外光谱分析。结果表明D301T离子交换树脂脱色率达到了55.67%,是本试验中绣球菌多糖脱色的最佳树脂。从绣球菌多糖中主要分离出两个组分,蒸馏水洗脱的组分(SCPl)和0.1%NaCl洗脱的组分(SCP2),SCP1与SCP2单糖组成都主要为甘露糖(Man)、葡萄糖(Glu)、半乳糖(Gla)和岩藻糖(Fuc),其摩尔比分别为0.043：0.06：1.75：0.386和0.0554：1.656：0.1249：0.0201。红外光谱分析得到其都具有多糖特征结构。采用体外抗氧化试验与建立衰老模型、灌胃不同浓度绣球菌多糖(SCP)进行体内抗氧化试验。结果表明在体外试验中绣球菌多糖(SCP)及其组分SCP1和SCP2对羟自由基、ABTS和DPPH均有很好的清除效果,同时具有很好的还原力,且SCP2作用最为突出。体内试验中,绣球菌多糖能够降低小鼠体内的MDA同时提高SOD与CAT含量,抗氧化效果较好。采用MTT法、倒置显微镜观察法和荧光染色法研究绣球菌多糖不同组分抑制人肝癌HepG-2细胞增殖作用。结果表明绣球菌多糖组分对人肝癌HepG-2细胞有较好的抑制作用,SCP2的抑制作用最好,其作用24h与48h的抑制率分别为48.12%和56.45%,倒置显微镜法与荧光染色法都能观察到绣球菌多糖具有使人肝癌HepG-2细胞凋亡的作用。建造小鼠肿瘤模型,灌胃不同浓度绣球菌多糖(SCP),测定小鼠肿瘤大小、脾脏与胸腺指数和血清中TNF-a与TNF-P含量,对肿瘤组织进行石蜡切片观察,同时对小鼠进行生存试验。结果表明绣球菌多糖对S180实体瘤小鼠的肿瘤有明显的抑制作用,绣球菌多糖高剂量组最高抑制率达到50%；对小鼠脾脏与胸腺指数均有提高作用,且能增加小鼠体内TNF-a与TNF-p的含量；石蜡切片观察肿瘤组织发现其呈现凋亡病理学特征；绣球菌多糖对小鼠生存寿命有一定延长作用,高剂量组对小鼠寿命延长32.6%。本研究表明,绣球菌多糖具有较高的生物活性,其中纯化的多糖SCP1是以半乳糖为主的多糖。SCP2是以葡萄糖为主的多糖。以葡萄糖为主的SCP2组分生物活性最高,且具有良好抗氧化功效,在体外对人肝癌HepG-2细胞的抑制作用也最好。绣球菌多糖在体内能抑制S180实体瘤小鼠的肿瘤和延长肿瘤小鼠生存寿命的抗肿瘤与作用抗氧化功效,是很值得重视和开发的药用真菌。
[Abstract]:The polysaccharide was extracted by water extraction, decolorized and deproteinized. The polysaccharide components were separated and purified. The monosaccharide composition of the polysaccharide was analyzed by infrared spectroscopy. The results showed that the decolorization rate of D301T ion exchange resin was 55.67, which was the best resin for decolorizing hydrangea polysaccharide. Two main components were isolated from the polysaccharides of Hydrangea, distilled water eluting components (SCPl) and 0.1%NaCl elution components (SCP2), and SCP1 and SCP2 monosaccharide groups in Chengdu were mainly mannose (Man), glucose (Glu),. The molar ratios of galactose (Gla) and fucose (Fuc), are 0.043: 0.06: 1.75: 0.386 and 0.0554: 1.656: 0.1249: 0.0201, respectively. Infrared spectrum analysis showed that all of them had the characteristic structure of polysaccharides. Antioxidation test in vitro and establishment of aging model were carried out in vivo with different concentrations of Hydrangea polysaccharide (SCP). The results showed that (SCP), its components SCP1 and SCP2 had good scavenging effect on hydroxyl radical, ABTS and DPPH in vitro, and had good reducing power, and SCP2 had the most prominent effect. In vivo experiment, Hydrangea polysaccharide could decrease MDA and increase the contents of SOD and CAT in mice, and the antioxidant effect was better. The effects of different components of hydrangea polysaccharides on the proliferation of human hepatoma HepG-2 cells were studied by MTT, inverted microscope and fluorescence staining. The results showed that the polysaccharides of Hydrangea had a better inhibitory effect on human hepatoma HepG-2 cells, and SCP2 had the best inhibitory effect. The inhibitory rates of SCP2 at 24h and 48h were 48.12% and 56.45%, respectively. It was observed by inverted microscope and fluorescence staining that Hydrangea polysaccharide could induce apoptosis of human hepatoma HepG-2 cells. The tumor model was established in mice. The tumor size, spleen and thymus index, TNF-a and TNF-P content in serum were measured by (SCP), with different concentrations of hydrangea polysaccharide. The tumor tissue was observed by paraffin section. At the same time, the survival test of mice was carried out. The results showed that Hydrangea polysaccharide had obvious inhibitory effect on tumor of S180 solid tumor mice, and the highest inhibitory rate of Hydrangea polysaccharide group was 50%. The indexes of spleen and thymus were increased, and the contents of TNF-a and TNF-p in mice were increased, and the pathological characteristics of apoptosis were found by paraffin section. Hydrangea polysaccharide could prolong the life span of mice to a certain extent, and the life span of mice in high dose group was increased by 32.6%. The results showed that the polysaccharides of Hydrangea had high bioactivity. The purified polysaccharide SCP1 was galactose and SCP2 was glucose. The SCP2 component with glucose as the main component has the highest bioactivity and has good antioxidant activity. It also has the best inhibitory effect on human hepatoma HepG-2 cells in vitro. Hydrangea polysaccharide can inhibit the tumor of S180 solid tumor mice in vivo and prolong the survival life of tumor mice. It is worthy of attention and development of medicinal fungi.
【学位授予单位】：福建师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R915

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