【作者】 李霞；

【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， 2008， 博士

【摘要】 本论文主要通过生物发酵技术培养黑盖木层孔菌;确定最优培养基;并且从菌丝体和发酵液中提取、纯化得到水溶性多糖;研究多糖的化学结构及抗肿瘤和免疫活性,并探讨多糖的结构与生物活性之间的关系。首先筛选了适宜黑盖木层孔菌菌丝体生长的碳源和氮源。最优碳源为可溶性淀粉,最优氮源为玉米面,菌丝的生长速度在有机氮源培养基上要明显优于无机氮源,确定蔗糖-玉米面为黑盖木层孔菌的最佳碳-氮源组合。并且,该菌种适宜液体发酵培养。黑盖木层孔菌经发酵培养后,得到发酵菌丝体,确定菌丝体多糖的最优提取条件为:提取温度100℃,浸提4次,每次2小时,浸提比1:30,各因素影响的大小次序是:提取次数>浸提比>浸提时间。分别从菌丝体和发酵液中分离得到水溶性粗多糖:菌丝体粗多糖PNW和胞外粗多糖PNM。GC分析粗多糖组成:PNW由甘露糖和葡萄糖构成,摩尔比为2.68:1.00;PNM由甘露糖、半乳糖和葡萄糖构成,摩尔比为2.02:1.00:3.21。采用冻融分级、脱蛋白、离子交换层析和分子筛凝胶层析等方法,分别从PNW和PNM中纯化出多糖PNW1和PNM1。HPLC检验PNW1和PNM1均为单一峰,平均分子量分别为33 kDa和29 kDa。GC分析,PNW1和PNM1均为杂多糖,由葡萄糖、半乳糖、甘露糖及少量的阿拉伯糖和岩藻糖组成,其摩尔比分别为3.26:8.77:6.44:1.00:1.35和20.06:8.72:6.94:1.00:0.76。采用部分酸水解、高碘酸氧化、Smith降解、甲基化等化学分析方法及IR、GC、GC-MS、13C NMR等仪器分析方法对PNW1和PNM1进行结构分析。确定了PNW1和PNM1的结构重复单元。PNW1的基本结构为:1→6- Glc和1→6- Gal构成主链,从Glc和Gal的O-2连接侧链,侧链由1→6- Man短链和Ara、Fuc、Man的末端构成。PNM1的基本结构为:1→6- Glc和1→6-Gal构成主链, Glc的O-2连接侧链,侧链由1→6- Man短链和Ara、Fuc、Glc、Man的末端构成。建立BALB/c小鼠体内S- 180实体瘤模型,进行体内抗肿瘤活性试验。经测定,菌丝体多糖和胞外多糖均能抑制小鼠体内移植性肿瘤S- 180的生长。并且,菌丝体多糖%的抗肿瘤活性优于胞外多糖。其中PNW1在400 mg/kg时抑瘤率最高,达到74.70%。在体外实验中,菌丝体多糖和胞外多糖对S- 180细胞没有的明显抑制作用。菌丝体多糖和胞外多糖均有免疫调节功能,能增加荷瘤小鼠的脾相关系数和胸腺相关系数,调节血清中的肿瘤坏死因子-α(TNF-α)的含量。多糖与脾淋巴细胞或巨噬细胞共培养后,表现出增强淋巴细胞的增殖和腹腔巨噬细胞的吞噬作用,并且促进巨噬细胞分泌NO和TNF-α。推测,其抗肿瘤作用是通过调节宿主的免疫机能实现的,可能与巨噬细胞有关。PNW1和PNM1的分子量相近,在30 kDa左右,均由葡萄糖、半乳糖、甘露糖及少量的阿拉伯糖和岩藻糖组成,各单糖的含量不同,结构也不同,但是他们的结构有共同的特点:主链是由1→6- Glc和1→6- Gal共同构成,由O-2连接侧链,侧链残基为1→6- Man短链。推测,分子量不高的带有O-2分支点的1→6真菌杂多糖具有抗肿瘤和免疫活性。比较两种多糖的活性,PNW1的抗肿瘤活性明显优于PNM1,免疫活性也优于PNM1,表明真菌多糖的抗肿瘤活性也与糖苷键的类型及其位置有关。更多还原

【Abstract】 The submerged culture medium of Phellinus nigricans and the extraction, purification, structure analysis and bioactivities of mycelium polysaccharide and medium polysaccharide were studied in this paper. The relationship between the structure and bioactivities of polysaccharides was also discussed.The effects of four kinds of carbon sources and nine kinds of nitrogen sources on the fungus Phellinus nigricans mycelia were studied. The results showed that the optimal carbon and nitrogen sources were starch and corn meal. The optimal combination was sucrose and corn meal. The optimal extraction conditions of polysaccharide from the mycelia of Phellinus nigricans were studied by orthogonal experiments. The optimal extracted conditions for hot water extraction were confirmed: the temperature 100℃, four times, two hours per times and the diffusion ratio 1:30. The effects of these influential factors on extracted efficiency were displayed as follows: extracted times > the diffusion ratio > extracted time.The two crude water-soluble polysaccharides, PNW and PNM, were isolated from the mycelia and its culture media of Phellinus nigricans, respectively. The results of the pharmaceutical experiments showed that oral administration of PNW and PNM inhibited the growth of transplant tumor of mice-transplanted Sarcoma 180 in vivo. Moreover, a higher inhibition ratio of PNW was obtained when compared with PNM. GC analysis indicated that PNW was composed of mannose and glucose with the molar ratio of 2.68:1.00, and PNM was composed of mannose, galactose and glucose with the molar ratio of 2.02:1.00:3.21.PNW1 and PNM1 were purified from PNW and PNM respectively using a combination of techniques such as frozen-thawed, deproteinization by Sevag method, anion-exchange chromatography on a column of DEAE-cellulose and gel filtration chromatography on a column of Sepharose CL-6B. PNW1 and PNM1 appeared as single symmetrical peak on HPLC, and the average molecular weights were 33 kDa and 29 kDa which were calculated through calculation using dextran standard. The results of monosaccharide composed analysis showed that glucose, galactose and mannose were the dominant monosaccharide units in the two polysaccharides and a small amount of arabinose and fucose were also existed; however, there was a difference in the corresponding molar ratios between PNW1 (3.26:8.77:6.44:1.00:1.35) and PNM1 (20.06:8.72:6.94:1.00:0.76).The major structural features of PNW1 and PNM1 were elucidated using partial acid hydrolysis, periodate oxidation, Smith degradation, 13C NMR, methylation and GC-MS. On the base of these results, the repeated units of PNW1 and PNM1 were established. On the base of the results obtained above, it was possible to conclude that the repeated unit of PNW1 contained a backbone composed of 1→6-linked-galactose and glucose, and 1→6-linkage-mannose branches attached to O-2 of galactose and glucose. PNM1 was composed of a backbone of 1→6-linked-galactose and glucose with 1→6- linkage- mannose branches attached to O-2 of glucose. The terminal monosaccharides were glucose, arabinose, fucose and mannose.At the dose of 100, 200, and 400mg/kg, both PNW1 and PNM1 exhibited anti-tumor activities against mice-transplanted Sarcoma 180 in vivo. PNW1 had better activity than PNM1 and the maximal tumor inhibition rate was 74.7% at the does of 400 mg/kg. While, no direct cytotoxic activity against Sarcoma 180 was observed in vitro. The significant increase in the relative spleen and thymus weight and expression of tumor necrosis factor-alpha (TNF-α) in serum was observed, that coincided with decreasing the tumor weight significantly. PNW1 and PNM1 could stimulate lymphocytes proliferation and increase production of nitric oxide (NO) and TNF-αin macrophages. The results indicate that both lymphocyte and macrophages were activated by polysaccharide from mycelium and culture medium of Phellinus nigricans. The anti-tumor effect of the polysaccharide was not directly tumoricidal but rather immunostimulating. PNW1 and PNM1 had the similar molecular weight of about 30 kDa and similar monosaccharide composition, but their molar ratios of monosaccharide were different. Their structures had common feature such as 1→6 linked backbone attached 1→6 mannose branches at O-2. All these results supposed that the fungus heteropolysaccharide with 1→6 linked backbone, O-2 branch and molecular weight of about 30 kDa had anti-tumor and immunomodulating activities. The differences between the two polysaccharides indicated that the type of sugar residue effects the anti-tumor activation of polysaccharide.更多还原