【作者】 杨焱；

【导师】 夏文水；

【作者基本信息】 江南大学 ， 食品科学， 2007， 博士

【摘要】 桑黄(Phellinus igniarius)是一种珍贵的药用真菌,含有多种化学成分,其中最主要的是多糖。近年来的研究表明,桑黄多糖具有抗肿瘤及其它多种生物功能,但目前的研究大多是用野生或发酵的桑黄提取物或粗多糖做研究对象,对人工栽培的桑黄及其多糖的结构和纯多糖活性的研究甚少。本论文对人工栽培桑黄中的多糖进行了分离纯化、结构鉴定及生物活性的系统研究,对开发利用我国的珍稀药用菌资源具有重要的学术意义,对桑黄天然抗肿瘤药物的开发也具有一定的应用价值。首先对桑黄子实体多糖进行了分离纯化,通过热水提取、乙醇分级沉淀分离得到粗多糖PIP30、PIP60和PIP80,通过超滤分离得到粗多糖PIWE1-10、PIWE10-100和PIWE100;6种粗多糖经体外刺激淋巴细胞增殖实验表明均有体外刺激淋巴细胞增殖的作用。PIP30经阴离子交换层析(DEAE-Sepharose Fast Flow)得到分级多糖P30U、P30W、P30S1和P30S2,PIP60经阴离子交换层析得到分级多糖P60U、P60W、P60S1和P60S2,PIWE1-10经阴离子交换层析得到分级多糖P1W、P1S1和P1S2,PIWE10-100经阴离子交换层析得到分级多糖P10W、P10S1和P10S2,PIWE100经阴离子交换层析得到分级多糖P100U、P100W、P100S1和P100S2;采用体外刺激淋巴细胞增殖实验对离子柱分级分离的多糖进行活性实验表明P30U、P30W、P30S1、P30S2、P60U、P60W、P60S2、P1S1、P1S2、P10S1、P10S2、P100U、P100W、P100S1和P100S2是有活性的。P60W、P60S1、P1S1、P10S1和P100S1经凝胶柱(Sephacryl S系列材料)层析获得五个纯多糖组分P60w1、P60s1、P1SP1、P10SP1和P100SP1。经高效液相色谱检测均为单一对称峰,说明五种多糖都是均一组分,紫外全扫描显示不含蛋白质和核酸特征吸收,红外光谱检测都含有糖的特征性基团,其相对分子量分别为1.71×10~4、2.07×10~4、1.48×10~4、2.20×10~4和2.56×10~4。其中P60w1、P1SP1、P10SP1和P100SP1有体外刺激淋巴细胞增殖作用的活性。运用离子色谱、气相色谱/质谱联用分析及相关的化学方法相结合对桑黄多糖中的单糖组分3-O-甲基-半乳糖进行了鉴定,发现桑黄Phellinus igniarius子实体分离的各纯多糖和发酵菌丝体多糖的糖组成中均包含3-O-甲基-半乳糖,而在其它药用真菌中尚无此单糖组成的报道,说明它可能是桑黄菌Phellinus igniarius多糖中含有的一种不同于其它药用真菌的特征性单糖。通过单糖组成分析、甲基化分析、核磁共振(1H NMR,13C NMR, 1H -1H COSY谱、TOCSY谱、HMQC谱、NOESY谱和HMBC谱)技术,对分离纯化得到的5个均一多糖的糖残基个数、糖残基的连接方式、构型、连接顺序等进行了研究。重点解析了活性较好、得率较高的均一多糖P60w1的结构,阐明P60w1的单糖组成有岩藻糖、半乳糖、葡萄糖、甘露糖和3-O-甲基-半乳糖,摩尔比为:1.12:2.26:1.00:1.03:1.11,主链主要是由α-(1→6)-D-半乳糖、α-(1→2,6)-3-O-甲基-D-半乳糖、β-(1→2,4)-3-O-甲基-D-半乳糖和α-(1→2,6)-D-甘露糖构成,侧链由α-端基L-岩藻糖和α-端基D-葡萄糖构成,其一级结构的重复单元如下式,目前尚无该多糖结构的研究报道。对其它四种均一多糖的结构分析表明,其糖组成中均包含岩藻糖、3-O-甲基-半乳糖、半乳糖、葡萄糖、阿拉伯糖、木糖和甘露糖,其中3-O-甲基-半乳糖和半乳糖占的比例较大,可能存在于主链上,木糖和阿拉伯糖占的比例较小;半乳糖有1,6和1,2,6两种连接方式,以1,6连接为主,岩藻糖主要以端基的形式存在;组成几种均一多糖的单糖的比例不同,葡萄糖和甘露糖在不同的多糖中连接方式有所不同。对桑黄醇提物PIEE、粗多糖PIP60、离子柱分离多糖P60S2和纯多糖P60w1进行了体外和体内的抗肿瘤实验研究,结果发现PIEE、PIP60、P60S2和P60w1对小鼠肝癌H22均有显著抑制作用,其中纯多糖P60w1的效果最好,优于阳性对照云芝糖肽;P60w1和P60S2对小鼠Lewis肺癌均有抑制作用,效果均优于阳性对照云芝糖肽,有效作用剂量为100 mg/(kg·d)。通过体外抑制肿瘤细胞生长试验研究发现,桑黄纯多糖在体外无抑制肿瘤细胞生长的作用,结合体外免疫活性研究结果,初步分析桑黄多糖的抗肿瘤作用可能是通过增强机体的免疫力来实现,而非通过直接杀伤肿瘤细胞起到抗肿瘤的作用。更多还原

【Abstract】 Phellinus igniarius (L.: Fr) Quel, is rare medicinal fungi contained polysaccharides besides many other kind of compounds. Polysaccharides of Phellinus igniarius has been reported to possess antitumor activities and inhibit tumor recrudescence and metastasis. The chemical composition and biological activities of crude polysaccharides of Phellinus sp. have been investigated and there is no reports relating to the chemical structure about the pure polysaccharides isolated from cultivated P. igniarius. In order to use this natural antitumor resource, the extraction, isolation, purification, structure characterization and pharmacological tests of the polysaccharides from the cultivated fruiting bodies of P. igniarius were carried out. It will be of scientific significance in researching and developing rare medicial fungi and be useful for searching new natural antitumor medicine.The crude polysaccharides of P. igniarius PIP30、PIP60、PIP80 and PIWE1-10、PIWE10-100、PIWE100 were obtained by ethanol precipitation and ultrafiltrate respectively after hot water extracting. P30U、P30W、P30S1 and P30S2 were isolated from PIP30, and P60U、P60W、P60S1 and P60S2 were isolated from PIP60, and P1W、P1S1 and P1S2 were isolated from PIWE1-10, and P10W、P10S1 and P10S2 were isolated from PIWE10-100, and P100U、P100W、P100S1 and P100S2 were isolated from PIWE100 using DEAE Sepharose F. F. The proliferation rates of lymphocyte in vitro of each polysaccharide fractions obtained during the isolation and purification were detected in order to observe their immune activities. The results showed that crude polysaccharides both obtained from ethanol precipitation and ultrafiltrate had the promoting effects on lymphocyte proliferation in various degrees. P30U、P30W、P30S1、P30S2、P60U、P60W、P60S2、P1S1、P1S2、P10S1、P10S2、P100U、P100W、P100S1 and P100S2 had the promoting effects on lymphocyte proliferation. P60W、P60S1、P1S1、P10S1 and P100S1 were further purified by gel-filtration chromatography (Sephacryl S-100-500 ) repeatedly. Five water-soluble homogeneous polysaccharides (P60w1、P60s1、P1SP1、P10SP1and P100SP1) were obtained. Lack of absorption at 280 nm and 260 nm by UV scanning indicated that contained no protein and nucleic acid. IR spectra showed that the sugar characteristic groups were existed. HPLC produced a single symmetrical peak, indicated homogeneity and their relatively molecular weigh were 1.71×10~4 Da、2.07×10~4 Da、1.48×10~4 Da、2.20×10~4 Da and 2.56×10~4 Da respectively. P60w1、P1SP1、P10SP1 and P100SP1 had the promoting effects on lymphocyte proliferation.A special monosaccharide 3-O-methylgalactose was identified by high performance anion exchange chromatography (HPAEC) and GC/MC. This monosaccharide existed both in fruiting body and mycelia polysaccharides of P. igniarius further indicate that it was possibly characteristic sugar in P. igniarius polysaccharides.The structures of five homogeneous polysaccharides were investigated using chemical (Sugar analysis, Methylation analysis, etc.) and spectroscopic methods (1D NMR: 1H NMR, 13C NMR,DEPT spectra; 2D NMR: 1H-1H COSY, TOCSY, HMQC, NOESY and HMBC spectra; etc.) to confirm the sugar composition, the number of sugar residues, the form of glycosyl linkage, the configuration of anomeric center, the sequence of sugar residues. The structure of P60w1 with high immune activity in vitro was elucidated in detail. Sugar analysis showed this polysaccharide was composed of fucose, galactose, glucose, mannose and 3-O-Me-galactose in the ratio of 1.12:2.26:1.00:1.03:1.11. Its main chain was composed ofα-D-1,6 linked galactosyl,α-D-1,2,6 linked 3-O-Me-galactosyl,β-D-1,2,4 linked 3-O-Me-galactosyl andα-D-1,2,6 linked mannose, side chain was composed of terminalα-L-fucose andα-D-glucose. The proposal structure is as follows:Sugar analysis showed other four polysaccharides were composed of fucose, 3-O-Me-galactose, galactose, xylose, mannose, glucose and a minor of arabinose, and the ratios of galactose and 3-O-Me-galactose were high, and the ratio of each sugar differed from each polysaccharide. Methylation analysis showed galactose and 3-O-Me-galactose were mainly 1,6 and 1,2,6 terminal linkage, fucose was terminal linkage, mannose and glucose had the different linkage in these polysaccharides.Antitumor experiments in mouse model with ethanol extract PIEE, crude polysaccharides PIP60, isolated polysaccharide P60S2 and pure polysaccharide P60w1 showed that PIEE, PIP60, P60S2 and P60w1 all had the inhibit effects on mouse liver cancer H22, and the effect of P60w1 was better than crude polysaccharides and positive control. P60w1and P60S2 had the inhibit effects on mouse Lewis lung cancer at the dosage 100 mg/(kg·d). Pure polysaccharides inhibiting tumor effect may have no relation to inhibiting tumor cell proliferation and potentially inhibited tumor by regulating immune function of body.更多还原