褐藻多糖硫酸酯免疫调节和抗肿瘤活性研究

目的研究褐藻多糖硫酸酯(Fucoidan)体外抗肿瘤及免疫调节作用。方法采用MTT法检测Fu-coidan对Hca-F肝癌细胞体外生长和对正常小鼠脾淋巴细胞增殖的影响;中性红比色法检测Fucoidan对小鼠脾Mφ吞噬活性的影响;ELISA法检测Fucoidan对脾细胞细胞因子分泌水平的影响。结果浓度低于1 000μg/ml时Fu-coidan对Hca-F肝癌细胞体外生长抑制作用不明显(P0.05);Fucoidan对脾淋巴细胞增殖、Mφ吞噬活性及细胞因子IL-6、IL-10、IL-12、IL-18和TNF-α的分泌均有增加趋势。结论 Fucoidan对体外培养的小鼠Hca-F肝癌细胞生长有一定的抑制作用;可提高细胞与分子免疫应答水平,调节细胞因子分泌,发挥抗肿瘤作用。     

Monthly changes in the content and monosaccharide composition of fucoidan from <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> (Laminariales,Phaeophyta)

Crude fucoidan was extracted from the brown alga Undaria pinnatifida collected monthly from April to last July in Peter the Great Bay (Japan Sea, Russia). The amount of crude fucoidan rose markedly from April to June–July (from 3.2 to 16.0% dry weight) as the plant matures. An analysis of the monosaccharide composition of the fucoidan extracted showed that the alga synthesized polysaccharides with various structures which were dependent on the algae age. In juvenile plants collected in April–May, this was represented by sulfated manno-galactofucan containing up to 19–28 mol% of mannose and about 20 mol% of galactose, whereas in matured plants collected in June–July, the polysaccharide was represented by a sulfated galactofucan containing more than 38 mol% galactose. It is postulated that the production of sori causes a subsequent effect on fucoidan synthesis and leads to an enhanced of crude fucoidan content and an increased molar concentration of galactose. Crude fucoidan content in sporophylls increased 5 times, and galactose content in this polysaccharide rose s1.6 times with sori formation. The structural characteristics of the fucoidan extracted from sporophylls of Undaria collected in July were also studied. The fractionation of crude fucoidan on DEAE-Sephadex A-25 gave two fractions, F1 and F2 in equal quantities. F1 was characterized as manno-galactofucan sulfate and F2 was galactofucan sulfate. The molecular weights of both fractions were in a range of 30–80 kDa. Analysis of fucoidan structure using ESI-FTICR mass spectrometry showed the presence of mixed oligosaccharides consisting of fucose and galactose. Presumably, the polysaccharide molecules contain blocks built up of successively linked residues of fucose and galactose. These blocks are built from two to five or more residues of monosaccharides. According to IR-spectroscopy data, the main portion of sulfates is located at C2; in addition, sulfate esters are also present at C4 on the fucose and C3 and C6 of the galactose units.     

Fucoidan对巨噬细胞RAW264.7的免疫调节作用

目的 观察褐藻多糖硫酸酯（Fucoidan）对巨噬细胞RAW264.7体外吞噬活性、细胞因子TNF-α和IL-6分泌,以及Toll样受体4（TLR4）mRNA表达的影响。方法 实验分对照组,Fucoidan高、中、低剂量组（浓度分别是200、400和800 μg/mL）。药物处理6~48 h后,MTT法检测RAW264.7细胞活力;中性红比色法检测细胞吞噬活性;ELISA法检测培养上清中TNF-α和IL-6的分泌水平;实时定量PCR检测Toll样受体4（TLR4）mRNA表达量。结果 与对照组相比,Fucoidan显著增强RAW264.7细胞代谢活力和吞噬能力（P＜0.01）,增加TNF-α和IL-6的分泌,上调TLR4的表达,呈剂量依赖关系。结论 Fucoidan可上调TLR4表达,增强巨噬细胞代谢和吞噬活性,增加TNF-α和IL-6的分泌,具有潜在的调节免疫作用。     

Isolation and characterization of marine bacterial strain degrading fucoidan from Korean Undaria pinnatifida Sporophylls

In spite of an increasing interest in fucoidans as biologically active compounds, no convenient commercial sources with fucoidanase activity are yet available. A marine bacterial strain that showed confluent growth on a minimal medium containing fucoidan, prepared from Korean Undaria pinnatifida sporophylls, as the sole carbon source was isolated and identified based on a 16S rDNA sequence analysis as a strain of Sphingomonas paucimobilis, and named Sphingomonas paucimobilis PF-1. The strain depolymerized fucoidan into more than 7 distinct lowmolecular- mass fucose-containing oligosaccharides, ranging from 305 to 3,749 Da. The enzyme activity was shown to be associated with the whole cell, suggesting the possibility of a surface display of the enzyme. However, a whole-cell enzyme preparation neither released the monomer Lfucose from the fucoidan nor hydrolyzed the chromogenic substrate p-nitrophenyl-alpha-L-fucoside, indicating that the enzyme may be an endo-acting fucoidanase rather than an alpha-L-fucosidase. Therefore, this would appear to be the first report on fucoidanolytic activity by a Sphingomonas species and also the first report on the enzymatic degradation of the Korean Undaria pinnatifida sporophyll fucoidan. Moreover, this enzyme activity may be very useful for structural analyses of fucose-containing polysaccharides and the production of bioactive fucooligosaccharides.     

The enhancing effect of fucoidan derived from Undaria pinnatifida on immunoglobulin production by mouse spleen lymphocytes

In this study, we revealed that a Mekabu (Udaria pinnantifida) extract enhanced immunoglobulin (Ig) production of mouse spleen lymphocytes. Furthermore, it was suggested that water-soluble and high molecular weight ingredients in the Mekabu extract have significant enhancing effect on Ig production. Therefore, fucoidan was estimated as the active component.     

Anti-influenza A virus characteristics of a fucoidan from sporophyll of Undaria pinnatifida in mice with normal and compromised immunity

Undaria pinnatifida, an edible brown alga, contains fucoidan (FuC), a sulfated polysaccharide, that inhibited the in vitro replication of influenza A virus, and stimulated both innate and adaptive immune defense functions in virus-infected mice. In the present study, the effects of oral administration of FuC were evaluated on influenza virus infection in immunocompetent and immunocompromised mice, where the efficacy of FuC was demonstrated in reducing viral replication, decreasing weight loss and mortality, and prolonging survival. Oral FuC resulted in increased neutralizing antibody production in the mucosa and blood. In contrast, while suppressing virus yields in mice more markedly than FuC, oseltamivir significantly reduced the neutralizing antibody titers in both the mucosa and blood. In immunocompromised mice, drug-resistant viruses frequently recovered after oseltamivir treatment; no resistant viruses were isolated from FuC-treated mice. FuC could be a candidate for the development of new therapeutic options including its combination with neuraminidase inhibitors such as oseltamivir.     

裙带菜假根中褐藻糖胶的提取

研究了温度、pH值、提取时间和水藻比及添加果胶酶和纤维素酶对采用乙醇沉淀法提取裙带菜假根中褐藻糖胶的影响。单因素和正交实验结果表明,80℃p、H 5、提取时间5 h下的褐藻糖胶得率最高,水藻比影响不明显。添加500 u/g果胶酶和纤维素酶可分别增加57%和46%。     

光同化产物在裙带菜（Undaria pinnatifida）中的运转

本文报告了用１４Ｃ标记的光同化产物在裙带菜［Ｕｎｄａｒｉａｐｉｎｎａｔｉｆｉｄａ（Ｈａｒｙ．）Ｓｕｒ］孢子体中的运转现象。观察到光同化产物约需２０ｍｉｎ才能从叶片表皮进入中肋的髓部。在自然条件下,光同化产物主要自叶片梢部经中肋向生长部运转,在叶片梢部和生长部之间存在明显的源一库关系。运转速度约为４２～４８ｃｍ／ｈ。用组织放射自显影的方法证实了髓部的喇叭丝是主要的运转组织。向下运转的溶于酒精的光同化产物中,甘露醇占５０％以上。还在光同化产物中观察到游离的谷氨酸、天门冬氨酸和丙氨酸。     

Extraction optimization and analysis of monosaccharide composition of fucoidan from Saccharina japonica by capillary zone electrophoresis

To optimize the extraction process of fucoidan from Saccharina japonica, an orthogonal test with four factors and three levels was designed in the present experiment. The results showed that the highest extraction yield was obtained under the following conditions: extraction temperature, 90 °C; extraction time, 1.5 h; number of extractions, 3; and pH of the extraction solution, 2.5. Furthermore, a capillary zone electrophoresis method using a UV detector was established for the analysis of the monosaccharide composition of fucoidan via 1-phenyl-3-methyl-5-pyrazolone derivatization. The results showed that fucoidans found in the three different samples all consisted of xylose, rhamnose, fucose, mannose, galactose, and glucuronic acid, while the mole ratios of monosaccharide components revealed differently. Moreover, fucose and galactose were found to be the most abundant monosaccharides in the samples. The above results could be further used for in-depth study on the mechanism of biological activity of fucoidan from S. japonica.     

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.     

裙带菜PSⅠ复合物的荧光特异性

以褐藻裙带菜(Undaria pinnatifida)为实验材料,采用蔗糖密度梯度超速离心的方法,去污剂SDS为增溶剂(SDS:Chi=20:1,4℃增溶20 min),蔗糖密度梯度为60%、50%、40%、30%、20%、15%和10%,分离制备光系统Ⅰ(PSⅠ)复合物.结果表明,40%蔗糖层带所含色素蛋白复合物是PS I复合物.利用红藻作参照对比,光谱结果表明从裙带菜中得到的PSⅠ复合物没有730 nm的荧光峰.分析认为这是所有褐藻包括裙带菜PSⅠ复合物的荧光特异性.     

Seasonal changes in growth rate, morphology and alginate content in Undaria pinnatifida at the northern limit in the Sea of Japan (Russia)

The growth, morphology, alginate yield and composition of Undaria pinnatifidawas studied from March to August in 2000 and 2001 at the northern limit of distribution of the species in Peter the Great Bay, the Sea of Japan (Russia). The changes in morphology, alginate yield and composition were caused by sporophyte growth and sporulation. The average rate of biomass change was 2–5% d⁻¹. The highest alginate content (51% d.wt) was obtained from blades, with lower values for sporophylls and midribs. An increase in alginate content was detected before sporulation. The conditions seem favourable for farming the alga in this region, with June the optimum month for harvesting. This revised version was published online in August 2006 with corrections to the Cover Date.     

褐藻裙带菜色素蛋白复合物的性质*

用去污剂DMG增溶褐藻裙带菜(Undaria pinnatifida)的类囊体膜,通过PAGE分离色素-蛋白复合物并分析其性质,结果表明:CPⅠa和CPⅠ都含有66kDa的多肽,低温荧光发射光谱中有715nm的长波荧光峰,激发光谱测定结果表明CPⅠa是含有墨角藻黄素的叶绿素a/c-蛋白复合物,CPⅠ是只含有叶绿素a的色素-蛋白复合物。CPa含有51、37、34和20kDa四种多肽,低温荧光发射峰位于683nm,激发光谱表明它含有叶绿素a、c和少量墨角藻黄素,是裙带菜的PSⅠ复合物。其余5条为捕光色素-蛋白复合物,它们都是由20kDa的多肽组成,其中LHC₁和LHC₃有相似的光谱特性,是墨角藻黄素-叶绿素a/c-蛋白复合物,LHC₂、LHC₄和LHC₅的光谱特性相似,是叶绿素a/c-蛋白复合物。     

Mucilages from the Ripe Fertile Frond of Undaria pinnatifida f. distans

The fractionation of the water extractable mucilage from the ripe fertile frond of Undaria pinnatifida f distans Miyabe et Okamura was studied. By an acid treatment, CPC-complex method, and DEAE-cellulose column chromatography this mucilage was separated into eleven fractions, of which the main polysaccharides were two fucogalactan sulfates (CPC-C-NaOH and CPC-N-NaOH) and an alginic acid. Judging by the ultracentrifugal analysis the formers are respectively composed of one component. They are probably identical, if their molecular sizes may differ slightly each other. Contents of the constituents in them are as follows: CPC-C-NaOH (main fucogalactan sulfate): fucose, 16; galactose, 21.5; sulfuric acid, 27.5%. CPC-N-NaOH (minor fucogalactan sulfate): fucose, 20.0; galactose, 30.7; sulfuric acid, 31.2%. Therefore, there exists in those compounds one sulfate residue per each sugar.

The ratio of d-mannuronic acid to l-guluronic acid in the alginic acid was found 0.87; and this value changed, by the KCI fractionation of it, to 1.64 for the KCI-insoluble fraction and to 0.76 for the KCI-soluble fraction.     

Molecular cloning and sequence analysis of methionine adenosyltransferase from the economic seaweed Undaria pinnatifida

The methionine adenosyltransferase gene (MAT) had been isolated from an economic seaweed Undaria pinnatifida by PCR using degenerate primers. The cDNA was 1,491 bp in length with an open reading frame of 1,194 nucleotides, encoding a deduced protein of 397 amino acids. The protein had a predicted molecular weight of 43.2 kDa, and the isoelectric point was 5.244. The sequence contains a 92 bp 5′-untranslated region (UTR) and a 205 bp 3′-UTR. The methionine adenosyltransferase (MAT) sequence of U. pinnatifida (UpMAT) shared 68–92 % identities with the previous published MAT sequences of other species. Phylogenetic analysis indicated that the phylogenetic relationship of UpMAT with some other seaweeds was closer than with those of higher plants. Under different stress conditions, the relative mRNA expression levels of the MAT of U. pinnatifida (UpMAT) were measured by real-time quantitative PCR, and the results demonstrated that the UpMAT might help to protect the alga against various abiotic stresses.     

Isolation and Nomenclature of Pigment-Protein Complexes from the Brown Alga (Undaria pinnatifida Harv.)

Eight kinds of pigment-protein complexes were resolved from the thylakoid membrane of the brown alga （Undaria pinnatifida Harv.) by using non-ionic detergent decanoyl-N-methylglucamide and PAGE technique. According to the apparent molecular weights, spectra characteristics, polypeptide compositions and referring to the higher plant spinach, eight pigment-protein complexes were named under Anderson′s terminology system as CPⅠa, CPⅠ, CPa, LHC1, LHC2, LHC3, LHC4, LHC5.     

褐藻裙带菜色素—蛋白质复合物的分离与命名

以非离子去污剂癸基－Ｎ－甲基葡萄糖胺为增溶剂,采用聚丙烯酰胺凝胶电泳技术从褐藻裙带菜（Ｕｎｄａｒｉａ ｐｉｎｎａｔｉｆｉｄａ Ｈａｒｖ．）的类囊体膜上分离到８种色素－蛋白质复合物。根据其表观分子量、光谱特性和多肽分析结果,并以高等植物菠菜（Ｓｐｉｎａｃｉａ ｏｌｅｒａｃｅａ Ｌ．）为对照,按照Ａｎｄｅｒｓｏｎ命名系统,８种色素－蛋白质复合物分别命名为ＣＰⅠ ａ、ＣＰⅠ、ＣＰａ、ＬＨＣ１、ＬＨＣ２、     

裙带菜同工酶酶谱特征及其遗传多样性的研究

用聚丙烯酰胺凝胶电泳（ＰＡＧＥ）技术对裙带菜丝状体青岛种群和舟山种群的１１种同工酶酶谱特征及其基因表达进行了研究,并作了遗传多样性的检测分析。结果表明：裙带菜丝状体青岛种群遗传多样性水平高于舟山种群：裙带菜丝状体种群还存在哑基因;另外,在裙带菜丝状体的乙醇脱氢酶酶谱中发现２个变异基因控制的２条酶带。     

Undaria pinnatifida exudates trigger shifts in seawater chemistry and microbial communities from Atlantic Patagonian coasts

Biological Invasions - The invasive kelp Undaria pinnatifida has spread from northeastern Asia to temperate coastal environments worldwide, with profound effects on colonized ecosystems. In this...