裙带菜褐藻糖胶的分离纯化及其结构分析

裙带菜经水提法提取得到褐藻糖胶粗多糖,经DEAE-Sepharose FF离子交换层析和 Sepharose 4B层析后,得到Sl、S2两个单一组分,相对分子质量分别为550 808、38 335.基本结构及单糖组成分析表明,二者均含有岩藻糖、糖醛酸、硫酸基、半乳糖、甘露糖、葡萄糖、鼠李糖、木糖、阿拉伯糖,但含量差别较大,推测与二者的生理活性的差异有关.     

羊栖菜中褐藻糖胶的组分分离及分析

为了确定羊栖菜褐藻糖胶中的活性组分 ,用分子量结合硫酸化程度的分级方法将其分成不同组分。经热水抽提的羊栖菜多糖 ,将去除褐藻胶、海带淀粉后剩余的褐藻糖胶经过DEAE—Sepharose离子交换色谱柱和Sepharose 4B凝胶层析柱被分成 5个褐藻糖胶组分。 5个组分中硫酸基和岩藻糖近似的分子数量之比分别为 1.863、0 .0 68、1.2 2 9、1.62 9,1.0 10 ;平均分子量分别为 2 .0× 10 4 、2 .2× 10 4 、8.2× 10 4 、2 1.4× 10 4 、32 .0× 10 4 ;百分含量分别为 8.6%、2 2 .9%、4 8.5 %、5 .7%、14.3%。     

褐藻多糖的分离提取及生理活性研究进展

褐藻多糖是海藻胶、褐藻糖胶和褐藻淀粉的统称,主要存在于褐藻中。褐藻糖胶作为其主要的生理活性物质,主要由L-岩藻糖和硫酸酯基组成,具有抗氧化、抗凝血、防癌抗肿瘤、抗病毒和消炎等活性。综述了褐藻多糖的提取分离方法和褐藻糖胶的生理活性研究进展,以期为褐藻多糖的应用提供参考。     

香菇多糖提取分离的研究

采用DEAE—cellulose柱层析和Sephadex G-200柱层析从香菇子实体热水浸提的粗多糖中分离出两个多糖组分Lenl—A、Len2-A,经旋光度法、Sepharose 4B柱层析、红外光谱等方法检测确定为均一多糖组分,且均为α-构型的吡喃糖,苯酚硫酸法测得其糖质量分数分别为91．5％和92．3％,旋光度为＋5．6°、＋11．2°,相对分子质量为375×10^3、718×10^3。     

褐藻糖胶的结构及构效关系研究

综述了褐藻糖胶结构方面的研究进展,以及褐藻糖胶的抗凝血活性和抗病毒活性与结构之间的关系。     

褐藻糖胶的化学组成及生物活性的研究

褐藻糖胶是一种从褐藻中提取的硫酸多糖,是一种天然活性物质,具有许多药用功能。近年来有关褐藻糖胶在医药方面的应用受到越来越多研究人员的关注。就其组成分析及其生物活性的研究作一综述。     

HILIC-ESI-HCD-MS/MS寡糖图谱分析用于海参褐藻糖胶的结构鉴定

褐藻糖胶也称为岩藻黄质、岩藻多糖、褐藻多糖,是一种硫化的多糖,常见于海参、海藻等褐藻纲及海藻类的植物中。研究发现,具有特定硫酸化结构的糖类结构域在生物功能中起着至关重要的作用。因此,对褐藻糖胶寡糖进行精细的结构表征,尤其是确定其硫酸化结构,对于理解褐藻糖胶的结构-功能关系至关重要。     

褐藻多糖及其衍生物的抗肿瘤作用

海洋植物作为一类有益健康的天然产品受到诸多关注。其中,褐藻科植物和褐藻的粗提取物在抗肿瘤活性方面显示出极大优势。鉴于此,褐藻粗提取物的活性成分褐藻多糖的抗肿瘤作用引起制药领域的极大兴趣。褐藻多糖包括褐藻胶、岩藻聚糖和褐藻淀粉等,主要来自海带、巨藻、泡叶藻、墨角藻等海藻植物。研究发现,褐藻糖胶的衍生物及海藻酸钠的衍生物具有降血压、调节免疫、抗炎抗病毒、抗氧化以及抗肿瘤的作用。本文介绍了褐藻多糖及其衍生物在抗肿瘤活性方面的研究现状。     

玉米芯木聚糖硫酸酯抗凝血活性及其机制的研究

采用活化部分凝血活酶时间(APTT)、凝血酶时间(TT)和凝血酶原时间(PT)检测了玉米芯木聚糖硫酸酯(wisX-SB)的抗凝活性,结果表明wisX-SB能明显延长APTT和TT,而不影响PT,提示wisX-SB是通过内源性和/或共同途径发挥抗凝血作用的。采用发色底物法及纤维蛋白原转化实验分别考察了wisX-SB对凝血酶及纤维蛋白原的作用,结果提示wisX-SB的抗凝机制包括:直接抑制纤维蛋白原的转化;通过增强抗凝血酶III(AT-III)的活性,抑制凝血酶活性,从而达到抗凝目的。较低浓度时以前者为主,较高浓度下两者皆起作用。     

野木瓜水溶性多糖的分离纯化及抗氧化活性研究(英文)

采用水提醇沉法获得了水溶性野木瓜粗多糖(CCCPs),经过脱蛋白、脱色及透析,得到了野木瓜精多糖(FCCPs),再经DEAE-纤维素和Sepharose CL-6B柱层析得到均多糖组分(CCP1)。高效液相色谱法测定CCP1的单糖组成为:鼠李糖、阿拉伯糖、果糖、甘露糖、葡萄糖,其摩尔比为0.034∶0.228∶0.045∶0.055∶0.638。体外抗氧化实验结果表明:不同纯度的野木瓜多糖都具有一定的抗氧化活性,并且随着多糖浓度增加,抗氧化活性增强,野木瓜粗多糖(CCCPs)对·OH和O-·2的清除能力比精多糖(FCCPs)和均多糖(CCP1)强。     

Structural investigation of a fucoidan containing a fucose-free core from the brown seaweed, Hizikia fusiforme

A fucoidan, obtained from the hot-water extract of the brown seaweed, Hizikia fusiforme, was separated into five fractions by DEAE Sepharose CL-6B and Sepharose CL-6B column chromatography. All five fractions contained predominantly fucose, mannose and galactose and also contained sulfate groups and uronic acid. The fucoidans had MWs from 25 to 950 kDa. The structure of fraction F32 was investigated by desulfation, carboxyl-group reduction, partial hydrolysis, methylation analysis and NMR spectroscopy. The results showed that the sugar composition of F32 was mainly fucose, galactose, mannose, xylose and glucuronic acid; sulfate was 21.8%, and the MW was 92.7 kDa. The core of F32 was mainly composed of alternating units of -->2)-alpha-D-Man(1--> and -->4)-beta-D-GlcA(1-->, with a minor portion of -->4)-beta-D-Gal(1--> units. The branch points were at C-3 of -->2)-Man-(1-->, C-2 of -->4)-Gal-(1--> and C-2 of -->6)-Gal-(1-->. About two-thirds of the fucose units were at the nonreducing ends, and the remainder were (1-->4)-, (1-->3)- and (1-->2)-linked. About two-thirds of xylose units were at the nonreducing ends, and the remainder were (1-->4)-linked. Most of the mannose units were (1-->2)-linked, and two-thirds of them had a branch at C-3. Galactose was mainly (1-->6)-linked. The absolute configurations of the sugar residues were alpha-D-Manp, alpha-L-Fucp, alpha-D-Xylp, beta-D-Galp and beta-D-GlcpA. Sulfate groups in F32 were at C-6 of -->2,3)-Man-(1-->, C-4 and C-6 of -->2)-Man-(1-->, C-3 of -->6)-Gal-(1-->, C-2, C-3 or C-4 of fucose, while some fucose had two sulfate groups. There were no sulfate groups in either the GlcA or xylose residues.     

香菇蛋白的分级纯化和结构分析

香菇粉经10℃pH 10的水提取制备香菇蛋白,得率13.1%,其蛋白含量47.5%,多糖含量24.2%.香菇蛋白经DEAE Sepharose CL-6B柱层析分级得5个级分,收集级分F1、F2、F3、F4,它们都是由蛋白和多糖构成的复合物.Sepharose CL-6B凝胶色谱显示,F2和F4的分子量分布较为均匀,且以蛋白为主,多糖含量很低;F3主要由两个分子量不同的蛋白级分构成,含有一定的多糖;F1中多糖含量较高,蛋白含量较少,且多糖分子量分布均匀.香菇蛋白的分子量主要集中在20 kDa～40 kDa之间.F1、F3、F4都属于酸性蛋白质,含有除色氨酸之外的7种必需氨基酸,除蛋氨酸含量较低外,其余必需氮基酸含量接近,且赖氨酸含量较高.红外光谱分析表明,香菇蛋白的二级结构主要为α-螺旋和无规卷曲.     

Production and purification of L-phenylalanine oxidase from Morganella morganii.

An enzyme fraction, acting predominantly on L-phenylalanine has been purified and characterized from Morganella morganii. The total envelope was prepared by disrupting the cells with a French press followed by high speed centrifugation. After solubilization of the particulate fraction with 0.1% Triton X-100 and then centrifugation, the resulting supernatant was layered onto a DEAE-Cellulose column. Active fractions eluted were applied to a Phenyl-Sepharose CL-4B column as the final purification step. The activity of the purified enzyme to various L-amino acids in decreasing order was phenylalanine, methionine, leucine, tryptophan, and to a much lesser extent cysteine and tyrosine. At 4 degrees C in 20 mM phosphate buffer pH 7.5, the partially purified fractions collected from the DEAE-Cellulose column were stable for 120 h. On the other hand, the purified fractions obtained from the Phenyl Sepharose CL-4B column showed a drastic decrease in activity within only 24 h. Mg2+ (up to 40 mM), Mn2+ or Ca2+ (up to 10 mM) stimulated the oxidation of the purified enzyme but increases beyond such levels decreased the enzyme activity. Co2+ (0.05 mM), Cu2+ (0.5 mM) or Zn2+ (0.1 mM) decreased the enzyme activity 37, 33 and 20%, respectively.     

Anticoagulant properties of sulphated polysaccharides and a proteoglycan from Codium fragile ssp. atlanticum

A high molecular weight sulphated (18.4%) proteoglycan was isolated from extracts of Codium fragile ssp. atlanticum by molecular exclusion chromatography on Sepharose 2B. Ion exchange chromatography, using Sepharose CL-6B, of lower molecular weight components eluted from the Sepharose 2B column gave two major products with sulphate contents of 10.2% and 7.5%, respectively. Anticoagulant activities of each of the three products were assessed using coagulation techniques and chromogenic substrate assays. An increase in anticoagulant effect was demonstrated by increasing concentration and sulphate content of each algal component. The mechanism of anticoagulant action was shown to be, principally, anti-thrombin in character due to potentiation of heparin cofactor II and antithrombin III activity. Although the anticoagulant substances described are unlikely to be used as antithrombotic therapeutic agents, they have uses as biomedical reagents for investigation of the processes of thrombin inhibition.     

Proteolytic fragmentation and peptide mapping of human carboxyamidomethylated tracheobronchial mucin

Human tracheobronchial mucin was isolated from lung mucosal gel by chromatography on Sepharose 4B in the presence of dissociating and reducing agents, and its thiol residues were carboxyamidomethylated with iodo[1(-14)C]acetamide. The 14C-carboxyamido-methylated mucin was purified by chromatography on Sepharose 2B. No low molecular weight components were detected by molecular sieve chromatography or polyacrylamide gel electrophoresis in the presence of dissociating and reducing agents or by analytical density centrifugation in CsCl/guanidinium chloride. After digestion of the purified 14C-mucin with trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone, three fractions (TR-1, TR-2, and TR-3) were observed by chromatography on Sepharose 4B. TR-1, a 260-kDa mucin glycopeptide fragment, contained all of the neutral hexose and blood group activity and 20% of the radioactivity in the undigested mucin. TR-1 was refractory to a second incubation with trypsin but could be digested by papain or Pronase to a smaller mucin glycopeptide fraction, as judged by the slight decrease in apparent molecular weight on Sepharose CL-4B. These mucin glycopeptides contained approximately 50% of the radioactivity in the TR-1 fraction, indicating that the glycosylated domains of carboxyamidomethylated tracheobronchial mucin contained thiol residues. The remainder of the radioactivity from papain or Pronase digests of TR-1 eluted, like the TR-3 fractions, in the salt fraction on Sepharose CL-4B. Peptide mapping of the nonglycosylated TR-3 fraction by TLC and high voltage electrophoresis yielded six principal and several less intensely stained ninhydrin reactive components, with the radiolabel concentrated in one of the latter peptides. Peptide purification of the TR-3 fraction by high pressure liquid chromatography on a C18 reverse phase column demonstrated the presence of four major peptides, with TR-3A being the dominant component. The TR-3D peptide contained S-carboxy-aminomethylcysteine and had 69% sequence similarity to the sgs-7 salivary glue protein of Drosophila.     

Anticoagulant activity of sulfated polysaccharide isolated from fermented brown seaweed Sargassum fulvellum

A sulfated polysaccharide with anticoagulant properties was isolated from the fermented brown seaweed Sargassum fulvellum. Freeze-dried S. fulvellum was fermented in an incubator for 10^th week at 25°C to convert seaweed macromolecules into anticoagulant sulfated polysaccharides (ASP). Anticoagulant activity was determined by an activated partial thromboplastin time (APTT) test using citrated human blood plasma. The 8^th week S. fulvellum crude seaweed extract (SWE) exhibited the highest blood anticoagulant activity. Therefore, 8^th week crude SWE was used for purification of ASP by two steps; DEAE cellulose anion-exchange followed by Sepharose 4B chromatography. The isolated ASP showed a single spot on agarose gel electrophoresis, which confirmed the purification status of our ASP. Polyacrylamide gel electrophoresis (PAGE) analysis showed that the molecular mass of the purified ASP was between 8 and 20 kDa. Polysaccharide and sulfate concentrations of the purified ASP were 180 and 29.70 μg mL⁻¹ respectively. ASP recovery was 1.32% (w/w) from the crude polysaccharide applied to the DEAE column. Purified ASP had a pH of 3.86 and was considered an acidic polysaccharide. Moreover, both ASP and heparin showed a relative clotting factor of 27.47 at the concentrations of 180 and 60 μg mL⁻¹ respectively. Therefore, S. fulvellum ASP can be considered a weaker anticoagulant than heparin. Results of the APTT, PT, and TT clotting assays showed that ASP was able to inhibit both intrinsic and extrinsic blood coagulation pathways. Finally, this study established a feasible and simple experimental protocol to isolate anticoagulant from fermented seaweeds leading to potential further development of anticoagulant agent for the pharmaceutical industry.     

Characterization of a fucoidan from Lessonia vadosa (Phaeophyta) and its anticoagulant and elicitor properties

Blades of Lessonia vadosa (Phaeophyta) were extracted with 2% CaCl(2) solution, affording in 4.4% yield a polysaccharide which contained fucose and sulfate groups in the molar ratio 1.0:1.12. The high negative optical activity value ([alpha](D)(22)=-134.0 degrees ), FT-IR and NMR analysis suggest the presence of a fucoidan. (13)C NMR spectrum of the polysaccharide obtained by solvolytic desulfation of native fucoidan indicated the major presence of 1-->3 linked alpha-l-fucan. Depolymerization of the native fucoidan with H(2)O(2) in the presence of copper(II) acetate gave in 54.8% yield a fraction with 33.7% of sulfate content. The native fucoidan (MW 320,000) showed good anticoagulant activity whereas the radical depolymerized fraction (MW 32,000) presented a weak anticoagulant activity. These polysaccharides showed significant activation of phenylalanine-ammonia lyase (PAL), lipooxygenase (LOX) and glutathione-S-transferase (GST) defence enzyme activities in tobacco plants.     

A comparison of isometallothionein synthesis in rat liver after partial hepatectomy and parenteral zinc injection.

Proteoglycan aggregates free of non-aggregating proteoglycan have been prepared from the annuli fibrosi and nuclei pulposi of intervertebral discs of three human lumbar spines by extraction with 4M-guanidinium chloride, associative density gradient centrifugation, and chromatography on Sepharose CL-2B. The aggregate (A1-2B.V0) was subjected to dissociative density-gradient ultracentrifugation. Three proteins of Mr 38 900, 44 200 and 50 100 found in the fraction of low buoyant density (A1-2B.V0-D4) reacted with antibodies to link protein from newborn human articular cartilage. After reduction with mercaptoethanol, two proteins of Mr 43 000 and two of Mr 20 000 and 14 000 were seen. The A1-2B.V0-D4 fraction, labelled with 125I, coeluted with both hyaluronate and a hyaluronate oligosaccharide (HA14) on a Sepharose CL-2B column. HA10 and HA14 reduced the viscosity of A1 fractions; HA4, HA6 and HA8 did not. HA14 decreased the viscosity of disc proteoglycans less than it did that of bovine cartilage proteoglycans. Thus, although a link protein was present in human intervertebral disc, it stabilized proteoglycan aggregates less well than did the link protein from bovine nasal cartilage.     

Chemical modification,characterization and structure-anticoagulant activity relationships of Chinese lacquer polysaccharides

A natural lacquer polysaccharide with complex branches was separated into two fractions, LPH (MW 16.9x10(4)) and LPL (MW 6.85x10(4)). Results of 13C NMR and FT-IR indicated they had the same structure. The treatment of LPL with sodium periodate led to a partial cut-off of side chains with 4-O-methyl-D-glucuronic acid in the terminal. These polysaccharides were sulfated in the presence of Py*SO3/DMSO. Depending on the reaction conditions, the products showed a different degree of sulfation (DS) ranging from 0.57 to 1.57 and different molecular weights ranging from 1.71x10(4) to 3.49x10(4). FT-IR analysis showed the equatorial primary OH at O-6 and the axial secondary OH at O-4 were sulfated. Activated partial thromboplastin time (APTT), prothrombin time and thrombin time (TT) assays showed the sulfated polysaccharides could prolong APTT and TT, but not TP. These activities strongly depended on the DS, the molecular weights (MW) and the branching structure of polysaccharides. DS of above 0.8 was essential for anticoagulant activity. The anticoagulant activity increased with the DS and the molecular weights. The molecular weights played a more important role. The branching structure of polysaccharides increased the activities. In our studies, the sulfated polysaccharides with the DS of 1.15 and the highest MW of 3.49x10(4) had the best blood anticoagulant activities.     

Keratan-sulphate-containing proteoglycans in human osteochondrophytic spurs of the femoral head

Newly synthesized and endogenous proteoglycan was isolated from human femoral head osteochondrophytic spurs. 35SO4-containing keratan sulphate was measured by its susceptibility to endo-beta-D-galactosidase (keratanase) and comprised 15-17% of the two subpopulations of a proteoglycan monomer fraction (D1) resolved by Sepharose CL-2B chromatography (Kav (I), 0.22; (II), 0.78). The size of the newly synthesized keratan sulphate in these fractions was large (Mr greater than 7,000). The hydroxylamine cleavage product of a proteoglycan aggregate fraction (A1) which eluted in the void volume of a Sepharose CL-2B column was immunoreactive with an anti-keratan sulphate monoclonal antibody, 5-D-4. Unlike the proteoglycan aggregate A1 fraction from bovine nasal cartilage, immunoreactivity against 5-D-4 was also found in chromatographic fractions retarded by Sepharose CL-2B. These results lend additional support to our assertion that the osteophyte extracellular matrix consists of hyaline cartilage-type proteoglycan. Stimulation of osteophyte proliferation may be useful as a repair mechanism for resurfacing denuded areas of osteoarthritic femoral heads.