Structural characterization of sulfated galactofucan from Undaria pinnatifida and its effect on type 2 diabetic mice

A sulfated galactoftucan designated as UpG was obtained from the brown algae Undaria pinnatifida by calcium chloride extraction.Chemical analyses showed that UpG is composed of galactose and fucose at a high sulfation level.Low-molecular weight UpGP-0.5 was prepared from UpG through acid hydrolysis for structure characterization.The backbones of UpG are determined to beα-(1,3)-Fuc,α-(1,4)-Gal,α-(1,3)-Gal,andα-(1,6)-Gal by GC-MS,FT-IR,NMR,and LC-MS analyses.Sulfate groups are modified at C2 and/or C4 of fucose and C3 and/or C4 of galactose.UpG could partially lower blood sugar and serum lipid levels in type 2 diabetic mice.Moreover,UpG treatment regulates the abundance of some specific gut microbiota,such as enriching the abundance of Muribaculum and Christensenellaceae,and reducing that of Bilophila,Tannerellaceae,Candidatus Saccharimonas and Anaerotruncus.The findings characterized the detailed structure of a sulfated galactofucan and investigated its potential for the treatment of type 2 diabetes mellitus.

裙带菜(Undaria pinnatifida)色素蛋白复合物的分离及光谱特性的初步研究

以褐藻裙带菜 (Undariapinnatifida)为实验材料 ,采用蔗糖密度梯度超速离心的方法纯化类囊体膜 ;以去污剂SDS为增溶剂 (SDS∶Chl =2 0∶1 ,4℃增溶 2 0min) ,再用蔗糖密度为60 %、5 0 %、40 %、30 %、2 0 %、1 5 %和 1 0 %的梯度离心法 ,从裙带菜中成功地分离出 5条含色素的蛋白质复合物带。离心结果显示在离心管顶部没有任何颜色 ,表明经过SDS增溶没有产生游离色素 ,纯化的类囊体膜和 5条色素条带进行吸收光谱分析 ,显示了属于叶绿素a(Chl a)的吸收峰 :41 8、436和 667到 672nm。叶绿素c(Chl c)的吸收峰 :61 5和 62 0nm。荧光发射最高峰位于 682到 690nm的区域。荧光激发光谱显示有Chl a和Chl c的激发峰 ,40 %层带所含有的色素复合物只有属于Chl a的吸收峰。分析结果表明 :1 0 %、1 5 %层带所含有的色素复合物是褐藻的捕光色素复合物 ;2 0 %、30 %层带所含有的色素复合物是浅绿色的PSⅡ复合体 (仍然含有捕光色素 ) ,40 %层带所含有的色素复合物是深绿色PSⅠ复合体 ,5 0

STUDY ON GAMETOPHYTE VEGETATIVE GROWTH OF UNDARIA PINNATIFIDA AND ITS APPLICATIONS

When cultured under certain environmental coriditions (25℃, light intensity 80 μmol/m2 ·s, LD12/12, in enriched seawater medium with 7 × 10-4mol/L NO3,-N, 1.56× 10-4mol/L, PO4-P and supplementsof other elements like Mn, Fe, I, etc.), mate and female gametophytes of U pinnatifida keptgrowing vegetatively and propagated fast at average daily fresh weight increase rate of about 20%. Theempirical formula Gm=Go. 3m was established to estimate the output of vegeative gametophytes. Vigorousvegetative gametophyte cells began to form reproductive structures (oogonium and spermatangium, whenthe tmperature was lower than 25℃ and other environmental factors wrre kept optimal. The sufficientsupply of gametophyte cells provided enough seeds for raising Undaria sporelings on prodiction scale.Controlled cross-breeding experiments using selcted male and female gametophyte clones which increasetheir cell number by mitosis instead of meiosis were also carried out in vitro. Juvenile sporophytes fromthe

INTRASPECIFIC CROSSINGS OF UNDARIA PINNATIFIDA (HARV.) SUR.-A POSSIBLE TIME-SAVING WAY OF STRAIN SELECTION

Unicellular gametophytes of Undaria pinnatifida (Harv.) Sur. were isolated in Qingdao, P. R. China in April 1993 and in Tokushima, southem Japan in March 1995. Diferent intraspecific crossings by using unicellular male and female gametophytes were successfully undertaken in Sept. of 1995 in Qingdao.Sporophytes were transplanted to two different locations for open an cultivation. One was at Zhanqiao (ZQ) Bay where the water current was slower than that of another location - Taipingjiao(TPJ). A total of218 adult sporophytes were harvested on January 12 and 18 from TPJ and ZQ repectively. For eacncombination, 10 sporophytes un cultivated. Analysis of the mprpholgical characteristics of adult spprophytes indicated that the longest length between two bases of the serration of pinnate bldes (W2) is a morphological characteristic that can be transferred from the parent plant to the next generations regardless of environmental variations. There was evidence that W2 was apparently determined by sex-linked factors, i.e, by male parental grametophyte.. Sporophytes from certain crossing combinations showed more vigorous growth than those from other crossing combinations. It is therrfore possible to select grametophyte strains which can be used as parental gametophytes for the seeding production of sporophytes with more vigorous growth within shorter cultivation period. The morphology of hybrids from a Qingdao strain and a Tokushima cultivated strain resembled that of both parental plants in frond feaures (wrinkled or smooth) and W2. Sporophyll formation also varied with strains. The fact that adult sporophytes resulting from the same crossing combinations have identical morphological characteristics under the same environmental conditions indicates the possibility of a new way to select strains which are expected to be ideal for commerical production by purposely selecting, propagating, and seeding unicellular gametophytes for sporeing production through freeliving techniques of gametophytes.

Fucoxanthin Isolated from Undaria pinnatifida Can Interact with Escherichia coli and lactobacilli in the Intestine and Inhibit the Growth of Pathogenic Bacteria

Fucoxanthin is a xanthophyll-type carotenoid that provides many benefits to human health. However, the mechanism by which fucoxanthin interacts with microbes and inhibits pathogenic bacteria is unknown. In this study, we investigated the effects of fucoxanthin isolated from the edible seaweed Undaria pinnatifida on pathogenic bacteria Escherichia coli and lactobacilli both in vitro and in vivo. Fucoxanthin strongly inhibited the growth of Gram-positive pathogenic bacteria but was less effective against Gram-negative bacteria. Fucoxanthin extracted from the crude mixture had a recovery rate of 93.38% and a purity of 82.70%, which were higher than those of fucoxanthin extracted using a previous method. Fucoxanthin also promoted the growth of intestinal mi- crobes in mice. Fucoxanthinol, a metabolite of fucoxanthin, was generated in the culture media. Fucoxanthin can be deacetylated into fucoxanthinol not only by conventional digestive enzymes in the digestive tract, but also by E. coli and lactobacilli in the intestine. These results indicate that fucoxanthin interacts with and influences E. coli and lactobacilli in the intestine. Therefore, fucoxanthin isolated from Undaria pinnatifida possibly can be applied in human health maintenance.

Circadian rhythms in the growth and reproduction of the brown alga Undaria pinnatifida and gametogenesis under different photoperiods

Circadian growth rhythm of the juvenile sporophyte of the brown alga Undaria pinnatifida was measured with the computer-aided image analysis system in constant florescent white light under constant temperature （ 10 ℃ ）. The growth rhythm persisted for 4 d in constant light with a free-running period of 25.6 h. Egg release from filamentous gametophytes pre-cultured in the light - dark regime was evaluated for six consecutive days at fixed time intervals in constant white light and 12 h light per day. Egg release rhythm persisted for 3 d in both regimes, indicating the endogenous nature. Temporal scale of egg release and gametogenesis in 18, 16, 12 and 8 h light per day were evaluated respectively using vegetatively propagated filamentous gametophytes. Egg release occurred 2 h after the onset of dark phase and peaked at midnight. Evaluation of the rates of oogonium formation, egg release or fertilization revealed no significant differences in four light-dark regimes, indicating the great plasticity of sexual reproduction. No photoperiodic effect in gametogenesis in terms of oogonium formation and egg release was found, but fertilization in short days was significantly higher than in long days. Results of this investigation further confirmed the general occurrence of circadian rhythms in intertidal seaweed species.

裙带菜(Undaria pinnatifida)配子体对四种抗生素的敏感性研究

采用显微计数的方法获得潮霉素、氯霉素、卡那霉素、硫酸链霉素等抗生素对裙带菜雌、雄配子体的致死率,通过统计分析研究了雌雄配子体对这些抗生素的敏感性。结果表明,裙带菜雌雄配子体对潮霉素和氯霉素敏感,482.7μg/ml潮霉素分别在第9、10天使雄配子体、雌配子体全部致死;雌雄配子体对氯霉素的敏感性还存在明显的差异,第8天氯霉素对雌雄配子体的半致死剂量分别是218.78μg/ml和371.54μg/ml。配子体对卡那霉素,硫酸链霉素相对不敏感,6000kU/L卡那霉素和800μg/ml的硫酸链霉素作用10天后雌雄配子体均无明显死亡现象。潮霉素和氯霉素可以作为裙带菜配子体的筛选试剂。

IDENTIFICATION AND SEQUENCE OF A cDNA CLONE CORRESPONDING TO A GENE INVOLVED IN DEVELOPMENT OF UNDARIA PINNATIFIDA

During the induction of gamete-producing gametangia, induced gametophytes werecollected at 4 days intervals (0,4,8, 12 d) and total RNAs were isolated by CsCl gradient ultracentrifu-gation. Some stage-specific expressed mRNAs were identified by differential display of mRNAs from dif-ferent developing stages of the gametophytes. The cDNA of one specific mRNA was verified, cloned andsequenced. This gene was specifically expressed during 4 days of induction, and had partial homologoussequence with tobacco IAA-binding protein gene. It suggests that this cDNA may represent a gene whichis related to the LAA regulating function during the development of the gametophytes.

Central Composite Design for Optimized Extraction of Polysaccharides from Undaria pinnatifida

Response surface methodology was used to optimize the extraction conditions of polysaccharides from Undaria pinnatifida.The effects of three independent variables,extraction time,extraction temperature and ratio of water to raw material were investigated by a central composite design.The experimental data were fitted to a second-order polynomial equation via multiple regression analysis and also examined with the appropriate statistical methods.The adjusted coefficient of determination（R^2Adj） for the model was 0.9171.An optimum extraction yield of 30.78% was obtained when the extraction temperature was 95 °C,extraction time was 4.5 h,and the ratio of water to raw material was 35（mL/g）.Under these conditions,validation experiments were done and the mean extraction yield of polysaccharides was 30.79%,which was in good agreement with the predicted model value.

Zoospores of Undaria pinnatifida:their efficiency to attach under different water velocities and conjugation behavior during attachment

In the invading course of Undaria pinnatifida, zoospore attachment in a dynamically changed subtidal water environment is crucial for the establishment of a potential population in alien waters. Among many abiotic factors that may interfere with the attachment process, water velocity is the most important one. In this investigation, the effect of water velocity on zoospore attachment of U. pinnatifida was investigated in an artificially designed system. It was found that freshly released zoospores that were transported by water flowing at 0 -16 cm/s showed no difficulty in attaching the smooth surface. Zoospore attachment decreased at elevated water flowing rates. At 70 cm/s no spore attachment occurred. Spores that have settled on glass slide for up to 1 h could not be stripped away by flowing water at a rate of 129 cm/s, the same was true of the 20 d old filamentous gametophytes. It was found that more than 70% of free-swimming zoospores tended to settle down adjacent to the settled spores and formed conjugated clusters from two up to a few hundred cells in still culture.

裙带菜(Undaria pinnatifida)雌诱激素在有性生殖过程的作用及影响其分泌的环境条件

以大型经济褐藻裙带菜配子体为实验材料,采用活性检测法对雌诱激素在有性生殖过程中的作用进行了研究。结果表明,刚刚排出的雌配子分泌了一种或几种易挥发的激素类物质,这些激素类物质能够促进精子释放并吸引精子向其游动,完成受精作用。另外,通过显微计数释放精子数的方法对影响裙带菜雌诱激素分泌的培养时间和培养的光照强度、环境温度、光周期等进行了实验,对精卵结合过程中雌诱激素分泌的最佳条件进行了研究,发现雌配子体在发育培养6—8d时检测到雌诱激素活性,最佳的温度条件为19℃,在光强40—160μmol/(m2·s)范围均检测到雌诱激素活性,最佳的光周期为12h:12h。

Metals in New Zealand <i>Undaria pinnatifida</i>(Wakame)

Undaria pinnatifida, Wakame is a popular edible seaweed in its native Asia and was first recorded in New Zealand in Wellington Harbor in 1987. It is classified as an unwanted species under the Biosecurity Act 1993, but there is growing interest in harvesting this seaweed for human consumption. The aim of this study was to evaluate the concentrations of metals in U. pinnatifida from several locations (Marlborough Sounds and Wellington harbor) and across seasons. In brief, the highest monthly mean concentration of metals found in New Zealand wild U. pinnatifida was Ca (16.97g·kg-1), K (48.48 g·kg-1), Mg (9.47 g·kg-1), P (12.05 g·kg-1), Cr (1.04 mg·kg-1), Cu (3.78 mg·kg-1), Mn (14.61 mg·kg-1), Ni (2.78 mg·kg-1), Se (0.83 mg·kg-1), Zn (35.03 mg·kg-1), As (46.71 mg·kg-1), Cd (2.91 mg·kg-1), Hg (0.042 mg·kg-1) and Pb (0.31 mg·kg-1). These results showed that New Zealand U. pinnatifida is a good source of the nutritionally important minerals calcium, magnesium, potassium and phosphorus. They also contained trace amounts of minerals such as chromium, copper, manganese, nickel, selenium and zinc. Contaminants such as arsenic, cadmium, mercury and lead were found at very low, safe, levels.

裙带菜(Undaria pinnatifida)类囊体膜分离及光谱性质研究

利用CaCl2溶液浸泡和超声波粉碎细胞技术相结合,从裙带菜细胞中提取类囊体膜。采用蔗糖密度梯度超速离心的方法(蔗糖密度梯度为60%、50%、40%、30%、20%、15%和10%)纯化类囊体膜。结果表明纯化的类囊体膜位于50%的蔗糖密度梯度。并进行光谱性质分析,从吸收光谱中可以看出最高峰位于670nm,荧光发射光谱最高峰位于680nm,缺少高等植物具有的730nm特征峰。

裙带菜(Undaria pinnatifida)配子体对氨态氮和硝态氮的吸收利用比较

本文对裙带菜(Undaria pinnatifida)配子体丰富培养的氮源进行了研究,结果发现裙带菜配子体能够有效利用氨态氮和硝态氮进行扩增,在30μmol/L氮离子浓度条件下,裙带菜配子体的日生长率能达到25%,可以满足配子体快速扩增的需要。裙带菜配子体在同时含有硝态氮和氨态氮的培养液中培养时,同时吸收两种氮源,并没有出现相互抑制的现象;配子体对氨态氮的吸收速率略大于硝态氮的吸收;对裙带菜配子体的氮吸收速率进行了测定,吸收速率最高分别可达24.3μmol/(g·h)和20.73μmol/(g·h),吸收速率随着氮浓度的增加而升高。

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。

Spermatozoid life-span of two brown seaweeds, Saccharina japonica and Undaria pinnatifida, as measured by fertilization efficiency

During sexual reproduction of seaweeds, spermatozoid (sperm) discharge is triggered by chemical messengers (pheromones) released by the female gametes. The chemotactic ability of the sperm ensures fertilization success. Using unialgal male and female gametophyte material under designated standard gametogenesis testing (SGT) conditions, the potential life-span of the sperm of two seaweeds, Saccharina japonica and Undaria pinnatifida, was assessed by their ability to fertilize eggs. Results show that within 20-30 min after being discharged, sperm of both species could complete fertilization without an apparent decline in fertilization rate. Although fertilization rate 60-120 min after sperm discharge dropped significantly in both species, some sperm were viable enough to fertilize the eggs. In S.japonica, at 12°C, some sperm were able to fertilize eggs up to 12 h after discharge. In both species, egg discharge rates (EDR) in the male and female mixed positive controls were significantly higher than those of all the sperm-testing groups. Doubling the seeded male gametophytes of S. japonica in the SGT tests significantly increased the EDR, further confirming the effect of the presence of the male on the female in terms of facilitating egg discharge from oogonia.

Isolation of Protoplasts from Undaria pinnatifida by Alginate Lyase Digestion

The aim of this study is to isolate protoplasts from Undaria pinnatifida. Protoplasts of the alga were isolated enzymatically by using alginate lyase, which was prepared by fermenting culture of a strain Vibrio sp. 510. Monofacterial method was applied for optimizing digestion condition. The optimum condition for protoplast preparation is enzymatic digestion at 28 ℃ for 2 h using alginate lyase at the concentration of 213.36 U (8 mL) every 0.5 g fresh thalline with NaCl 50 and at the shaking speed of 150 r min -1 during digestion. The protoplast yield can reach 2.62±0.09 million per 0.5 g fresh leave under the optimum condition. The enzyme activity is inhibited by Ca 2+ and slightly enhanced by Fe 2+ and Mn 2+ at concentrations of 0.05, 0.08 and 0.10 mol L -1.

High genetic diversity in gametophyte clones of Undaria pinnatifida from Vladivostok, Dalian and Qingdao revealed using microsatellite analysis

Breeding practice for Undaria pinnatifida (Harvey) Suringar requires the screening of a large number of offspring from gametophyte crossings to obtain an elite variety for large-scale cultivation. To better understand the genetic relationships of different gametophyte cultures isolated from different sources, 20 microsatellite loci were screened and 53 gametophyte clone cultures analyzed for U. pinnatifida isolated from wild sporophytes in Vladivostok, Russia and from cultivated sporophytes from Dalian and Qingdao, China. One locus was abandoned because of poor amplification. At the sex-linked locus of Up-AC-2A8, 3 alleles were detected in 25 female gametophyte clones, with sizes ranging from 307 to 316 bp. At other loci, 3 to 7 alleles were detected with an average of 4.5 alleles per locus. The average number of alleles at each locus was 1.3 and 3.7 for Russian and Chinese gametophyte clones, respectively. The average gene diversity for Russian, Chinese, and for the combined total of gametophyte clones was 0.1, 0.4, and 0.5, respectively. Russian gametophyte clones had unique alleles at 7 out of the 19 loci. In cluster analysis, Russian and Chinese gametophyte clones were separated into two different groups according to genetic distance. Overall, high genetic diversity was detected in gametophyte clones isolated from the two countries. These gametophyte cultures were believed to be appropriate parental materials for conducting breeding programs in the future.

裙带菜多糖抑制肝癌细胞增殖和迁移的机制

目的探讨裙带菜多糖(sulfated polysaccharide from Undaria pinnatifida,SPUP)对肝癌细胞增殖、迁移的影响及潜在机制。方法MTT和克隆形成实验检测SPUP对肝癌细胞HepG2、SMMC-7721增殖的影响;Hoechst 33258染色和Western blot法检测SPUP干预后对HepG2、SMMC-7721细胞凋亡的影响;Transwell和划痕法检测HepG2、SMMC-7721细胞的迁移和侵袭;免疫荧光和Western blot法检测SPUP对肝癌细胞自噬、上皮-间质转化(epithelial-mesenchymal transition,EMT)和基质金属蛋白酶2(matrix metalloproteinase 2,MMP-2)、MMP-9蛋白的影响。结果SPUP对HepG2、SMMC-7721细胞有短期和长期的抑制增殖作用,对正常肝细胞L-02无明显抑制作用,SPUP可促进两种肝癌细胞凋亡和自噬水平。同时,SPUP可以抑制肝癌细胞的迁移侵袭能力,降低EMT和MMPs水平。结论SPUP通过促进凋亡和自噬、抑制EMT和MMPs,进而抑制肝癌细胞的增殖和迁移。