Antioxidant activity of different molecular weight sulfated polysaccharides from Ulva pertusa Kjellm (Chlorophyta)

Polysaccharides extracted from Ulva pertusa Kjellm ( Chlorophyta) are a group of sulfated heteropolysaccharides, the ulvans. In this study, different molecular weight ulvans were prepared by H2O2 degradation and their antioxidant activities investigated including superoxide and hydroxyl radical scavenging activity, reducing power and metal chelating ability. The molecular weights of natural and degraded ulvans were 151.7, 64.5, 58.0, and 28.2 kDa, respectively, as determined by high performance gel permeation chromatography. Among the four samples, U-3 ( the lowest molecular weight sample) showed significant inhibitory effects on superoxide and hydroxyl radicals with IC50 values of 22.1 mu g mL(-1) and 2.8 mg mL(-1); its reducing power and metal chelating ability were also the strongest among the four samples. All the other samples also demonstrated strong activity against superoxide radicals. The results indicated that molecular weight had a significant effect on the antioxidant activity of ulvan with low molecular weight ulvan having stronger antioxidant activity.

Antihyperlipidemic effects of different molecular weight sulfated polysaccharides from Ulva pertusa (Chlorophyta)

Ulvan, a sulfated polysaccharide from Ulva pertusa, was degraded to yield two low molecular weight fractions U1 and U2. The molecular weights of ulvan and its fractions were determined and varied from 151.6 to 28.2 kDa. They were fed to rats on a hypercholesterolemic diet for 21 days to evaluate and compare the antihyperlipidemic actions. Ulvan-based diet significantly lowered the levels of serum total cholesterol (-45.2%, P < 0.05) and low density lipoprotein cholesterol (LDL-cholesterol, -54.1%, P < 0.05). While U1- and U2-based diets significantly elevated the levels of serum high density lipoprotein cholesterol (HDL-cholesterol, +22.0% for U1, not significant; +61.0% for U2; P < 0.05) and reduced triglyceride (TG, -82.4% for U1, -77.7% for U2; P < 0.05) in rats as compared to control diet. In addition, consumptions of various ulvans significantly increased fecal bile acid excrement. The results indicated that ulvans with different molecular weights exhibited diverse effects on lipid metabolism. The high molecular weight ulvan was effective in serum total and LDL-cholesterol, whereas low molecular weight fractions were in TG and HDL-cholesterol. The fractions were considered to be more beneficial to hyperlipidemia associated with diabetes over ulvan. (C) 2003 Elsevier Ltd. All rights reserved.

Polysaccharides from Ulva pertusa (Chlorophyta) and preliminary studies on their antihyperlipidemia activity

Polysaccharides from Ulva pertusa were isolated and prepared by extraction in hot water and precipitation by ethanol. The water-soluble polysaccharides were chemically well defined, containing 47.0% total carbohydrate, 23.2% uronic acids, 17.1% sulfate groups, 1.0% N and 29.9% ash. Gas chromatography analysis demonstrated that the neutral sugars were mainly composed of rhamnose, xylose and glucose and smaller amounts of mannose, galactose and arabinose. The FTIR and C-13-NMR spectra indicated that basic repeating units of the polysaccharides were (beta-D-GlcpA-(1->4)-alpha-L-Rhap 3S) and (alpha-L-IdopA-(1->4)-alpha-L-Rhap 3S). Fifty ICR mice were used to study the effect of water-soluble polysaccharides from Ulva pertusa on the level of plasma lipids, with inositol niacinate as positive control. The results indicated that the polysaccharides significantly lowered the contents of plasma total cholesterol, low-density lipoprotein cholesterol, triglyceride and markedly increased the contents of serum high-density lipoprotein cholesterol, compared with the hyperlipidemia control group (p<0.01). Moreover, administration of polysaccharides significantly decreased the atherogenic index. The present results suggest that the polysaccharides from Ulva pertusa have great potential for preventing ischemic cardiovascular and cerebrovascular diseases.

Extension of life span and improvement of vitality of Drosophila melanogaster by long-term supplementation with different molecular weight polysaccharides from Porphyra haitanensis

Polysaccharides isolated from Porphyra (porphyran) have been known to have diverse biological activities, including immunomodulatory and antioxidant activities. The molecular weight-antiaging activity relationship of degraded porphyrans was examined in this study. Natural porphyran was extracted from P. haitanensis, and then was degraded into different molecular weight fractions, P1 molecular weight 49 kDa, P2 molecular weight 30 kDa, P3 molecular weight 8.2 kDa, by free radical. The influence on life span and vitality of porphyrans were carried out on Drosophila melanogaster. We found that all the degraded porphyrans and natural porphyran (P), added daily to the diet, can significantly increase the life span of D. melanogaster, except for P3. Among them, P1 exhibited the most prolonging life span activity. Furthermore, vitality of middle-aged flies (assessed by measuring their mating capacity) receiving porphyrans was increased considerably in comparison with the controls. Finally, in the heat-stress test, we observed a remarkable increase in survival time, especially in P3-diet groups. These results suggest that porphyrans may be effective in reducing the rate of the aging process and molecular weight has important influence on the effects. It seems that P1 and P2, possessed higher molecular weight, may be more useful in normal metabolic condition and P3, possessed the lowest molecular weight, may be more beneficial for D. melanogaster in stress condition. (C) 2007 Elsevier Ltd. All rights reserved.

Extraction of the polysaccharides from five algae and their potential antioxidant activity in vitro

The sulfated polysaccharides extracted from algae possess excellent antioxidant activities. In this study, we prepared five polysaccharides extracted from five algae including one brown alga Laminaria japonica. one red alga Porphyra haitanensis and three green algae Ulva pertusa, Enteromorpha linza and Bryopsis plumose. And then the antioxidant activities of all the samples were investigated including scavenging effects of superoxide and hydroxyl radicals, and reducing power. The chemical analysis and FT-IR spectrum showed these extracts were polysaccharides. And in addition, we found that certain polysaccharide exhibited stronger antioxidant activity in certain antioxidant activity. Factors effecting and attributing to radical scavenging effect need to be further studied. (C) 2010 Elsevier Ltd. All rights reserved.

白皮松花粉管发育过程中RNA、蛋白质和多糖类物质的合成及其动态变化

花粉管是有花植物受精过程中雄性生殖单位的载体，它同根毛、真菌菌丝一样，具有典型的极性顶端生长模式。裸子植物花粉与被子植物相比，具有萌发时间较长，生长缓慢等特点。但是目前人们对于裸子植物花粉萌发和花粉管生长的机理还不清楚。本文以裸子植物白皮松（Pinus bungeana）的花粉为材料，采用细胞学和生理生化方法，包括应用普通光学显微镜、荧光显微镜、激光扫描共聚焦显微镜、显微红外光谱（FTIR）和透射电镜（TEM）等技术，对其花粉萌发和花粉管生长过程进行了较为系统的研究，旨在进一步揭示裸子植物花粉管发育的调控机制。 本论文首先研究了外源Ca2+ 和3种调钙药物（A23187、EGTA、TMB8）对白皮松花粉萌发和花粉管生长的影响。结果表明，在离体培养条件下，高浓度的Ca2+（1%）能完全抑制白皮松花粉的萌发，低浓度的Ca2+ 则影响不大，而花粉萌发和花粉管生长的最适Ca2+ 浓度为0.01%。用Ca2+ 载体A23187、Ca2+ 螯合剂EGTA和钙通道阻滞剂TMB8分别处理花粉后，花粉萌发和花粉管生长均受到抑制。另外，用 Ca2+ 荧光探针 Fluo-3AM标记，对Ca2+ 的分布变化进行了观察，发现在花粉萌发的初期，Ca2+ 向萌发孔聚集。在正常生长的花粉管中Ca2+ 呈梯度分布，顶端荧光最强。与对照相比，A23187处理后花粉粒中荧光增强，而EGTA和TMB8处理的花粉粒中荧光强度均减弱。并且这3种调钙药物还破坏了花粉管顶端的Ca2+ 浓度梯度，最终导致花粉管的生长受阻。 花粉萌发和花粉管的生长依赖于RNA和蛋白质的不断合成。在放线菌素D的存在下，花粉萌发基本不受影响，但花粉管的生长速度下降，花粉管中RNA含量也减少。而经过放线菌酮处理后，花粉萌发和花粉管生长均受到抑制，花粉管中蛋白质含量降低，同时花粉管顶端显著膨大。通过SDS-PAGE的结果表明，花粉粒萌发前后蛋白质图谱有明显差异。FTIR光谱分析表明，两种抑制剂处理均导致花粉管壁的化学组成发生了变化，例如蛋白质和饱和酯含量减少，而羧酸的含量增加。此外，由放线菌酮和放线菌素D处理后，花粉管的超微结构也发生了明显变化，其中特别是花粉管顶端的分泌系统遭到严重破坏。 纤维素的正常合成对于白皮松花粉管的生长是必需的。在正常培养基中添加纤维素生物合成抑制剂2，6-二氯苯腈（DCB）后，花粉萌发几乎不受影响，但是花粉管的形态发生异常，生长速率降低。DCB处理还导致花粉管壁中纤维素含量下降，而胼胝质在花粉管顶端积累。用识别酯化果胶的JIM7和识别酸性果胶的JIM5对离体培养的白皮松花粉管进行标记后，发现果胶成分呈异常分布图式。FTIR光谱分析结果表明花粉管细胞壁中蛋白质、羧酸以及饱和酯含量增加。同时，在电镜下观察发现，花粉管细胞壁顶端呈现不均匀加厚，其中主要的细胞器，如高尔基体和线粒体等膜结构均遭到破坏。 上述结果说明，白皮松成熟花粉粒中已含有花粉萌发和花粉管早期生长所必需的Ca2+ 和RNA，但是在花粉管的后续伸长过程中仍需要外源Ca2+ 的参与以及新RNA、蛋白质的不断合成。与被子植物不同，裸子植物花粉萌发的启动也需要新蛋白的合成。尽管在花粉管中纤维素的含量很低，但是对于细胞壁的构建、花粉管的正常形态的维持起着关键作用。

Cytochemical changes in the developmental process of Nostoc sphaeroides (cyanobacterium)

There are several apparent developmental stages in the life cycle of Nostoc sphaeroides Kutzing, an edible cyanobacterium found mainly in paddy fields in central China. The cytochemical changes in developmental stages such as hormogonia, aseriate stage, filamentous stage and colony in N. sphaeroides were examined using fluorescent staining and colorimetric methods. The staining of acidic and sulfated polysaccharides increased with development when hormogonia were used as the starting point. Acidic polysaccharides (AP) were most abundant at the aseriate stage and then decreased, while the sulfated polysaccharides (SP) were highest at the colony stage. Quantitatively, along the developmental process from hormogonia to colony, total carbohydrates first increased, then became stable, and then reached their highest level at the colony stage, while reducing sugars were highest at the hormogonia stage and then decreased sharply once development began. SP were not detectable in the hot water soluble polysaccharides (HWSP), and hormogonia had the lowest content of AP, while old colonies had the highest. The AP content of the aseriate stage, filamentous stage and young colony stage were very similar. The evolutionary relationships reflected in the developmental stages of N. sphaeroides are discussed.

Purification and characterization of hydrosoluble components from the sap of Chinese lacquer tree Rhus vernicifera

Continuous gradient elution chromatography (CGEC) was employed to purify and separate enzymes and polysaccharides from the sap of Rhus vernicifera Chinese lacquer tree. There are three different molecules with laccase enzyme activity. Two are enzymes of each other (L1, and L2), whereas the third (RL) is an entirely separate entity. Two polysaccharides (GP1 and GP2) were also found. The Rhus laccase (RL), and isoenzymes L1 and L2, have peak molecular masses of 109,100, 120,000, 103,000 respectively; each has four copper atoms per molecule, and the pI values were 8.2, 8.6, and 9.1, respectively. The structure of the laccases was studied by Fourier-transform infrared (FT-IR) and Matrix-assisted laser desorption/ionization time-of flight (MALDI-TOF) mass spectrometry. The typical amide I (1646 cm(-1)) and amide II (1545 cm(-1)) bands were observed. The results from MALDI-TOF were similar to those from CGEC, but the molecular mass from the MALDI-TOF was significantly different from that obtained from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). (c) 2006 Elsevier B.V. All rights reserved.

Effect of dietary carbohydrate sources on growth performance and utilization for gibel carp (Carassius auratus gibelio) and Chinese longsnout catfish (Leiocassis longirostris Gunther)

The nutritional function of monosaccharides, disaccharides and polysaccharides for omnivorous gibel carp and carnivorous Chinese longsnout catfish were investigated and the ability of these two species to utilize carbohydrates was compared. For each species, triplicate groups of fish were assigned to each of five groups of isoenergetic and isonitrogenous experimental diets with different carbohydrate sources: glucose, sucrose, dextrin, soluble starch (acid-modified starch) and alpha-cellulose. The carbohydrates were included at 60 g kg(-1) in Chinese longsnout catfish diets and at 200 g kg(-1) in gibel carp diets. A growth trial was carried out in a recirculation system at 27.8 +/- 1.9 degrees C for 8 weeks. The results showed that fish with different food habits showed difference in the utilization of carbohydrate sources. For gibel carp, better specific growth rate (SGR) and feed efficiency (FE) were observed in fish fed diets containing soluble starch and cellulose, but for Chinese longsnout catfish, better SGR and FE were observed in fish fed diets containing dextrin and sucrose. Apparent digestibility coefficient of dry matter (ADC(d)) and apparent digestibility coefficient of energy (ADC(e)) were significantly affected by dietary carbohydrate sources in gibel carp. ADC(d) and ADC(e) significantly decreased as dietary carbohydrate complexity increased in Chinese longsnout catfish except that glucose diet had medium ADC(d) and ADC(e). In both species, no significant difference of apparent digestibility coefficient of protein was observed between different carbohydrate sources. Dietary carbohydrate sources significantly affected body composition, and liver phosphoenolpyruvate carboxykinase (PEPCK), pyruvate kinase (PK), glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME) activities also varied according to dietary carbohydrate complexity. Fish with different food habits showed different abilities to synthesize liver glycogen, and the liver glycogen content in gibel carp was significantly higher than in Chinese longsnout catfish. The influence of carbohydrate source on gluconeogenesis and lipogenesis was also different in the two fish species.

Salt tolerance of Microcoleus vaginatus Gom., a cyanobacterium isolated from desert algal crust, was enhanced by exogenous carbohydrates

Microcoleus vaginatus isolated from a desert algal crust of Shapotou was cultured in BG-11 medium containing 0.2mol l(-1) NaCl or 0.2mol l(-1) NaCl plus 100mg l(-1) sucrose, extracellular polymeric substances (EPS) or hot water-soluble polysaccharides (HWP), respectively. Photosynthetic oxygen evolution rates, photosystem 11 activity (Fv/Fm) and dark respiration of NaCl-stressed cells were enhanced significantly by the added sucrose or EPS under salt stress conditions (0.2mol l(-1) NaCl). Compared with cells treated with salt alone, sodium contents in cells reduced significantly; the content of cellular total carbohydrate did not change, and intracellular sucrose, water-soluble sugar increased significantly following the addition of exogenous carbohydrates. Sucrose synthase (SS) activity of NaCl-stressed cells increased following the addition of sucrose, and sucrose phosphate synthase (SPS) activity of NaCl-stressed cells increased following the addition of exogenous sucrose, EPS or HWP compared with cells stressed with NaCl only. The results suggested that the extruded EPS might be re-absorbed by cells of M. vaginatus as carbon source, they could increase salt tolerance of M. vaginatus through the changes of carbohydrate metabolism and the selective uptake of sodium ions. (C) 2003 Elsevier Science Ltd. All rights reserved.

A physico-chemical comparative study on extracellular carbohydrate polymers from five desert algae

Hydrodynamic properties of five newly isolated algal extracellular polysaccharides with putative adhesive properties are described, using a combination of size exclusion chromatography, total or 'multi-angle' laser light scattering and analytical ultracentrifugation. The respective polysaccharides had been extracted from four filamentous cyanobacteria: Microcoleus vaginatus, Scytonema javanicum, Phormidium tenue and Nostoc sp. and a coccoid single-cell green. algae Desmococcus olivaceus that had been separated from desert algal crusts of the Chinese Tegger Desert. SEC/MALLS experiments showed that the saccharides had, diverse-weight average molecular weights ranging from 4000 to 250,000 g/mol and all five showed either bi-modal or tri-modal molecular weight distribution profiles. Use of the Mark-Houwink-Kuhn-Sakurada (MHKS) scaling relationship between sedimentation coefficient and (weight average) molecular weight for the five samples, assuming a homologous conformation series revealed an MHKS b exponent of (0.33 +/- 0.04), suggesting a conformation between that of a stiff rod (b similar to 0.18) and a random coil (b similar to 0.4-0.5), i.e. a 'flexible rod' or 'stiff coil'. (C) 2003 Elsevier Ltd. All rights reserved.

西藏胡黄连和鸡矢藤的化学成分及黄芪多糖的提取工艺研究

本论文由四部分组成，前三部分为实验论文，第四部分为文献综述。第一、二部分分别报道了中药西藏胡黄连和鸡矢藤的化学成分研究结果。从两种药用植物中共分离和鉴定了32个化学成分，其中3个为新化合物。第三部分为黄芪多糖的提取工艺研究。第四部分概述了近年来植物多糖的研究进展。 第一章为西藏胡黄连化学成分研究。通过正、反相硅胶柱层析等分离方法从药用植物西藏胡黄连（Picrorhiza scrophulariiflora Pennell）的根茎中共分离纯化出7个化合物。运用MS、1H-NMR、13C-NMR、DEPT、HSQC和HMBC等现代谱学方法，结合理化分析对这些化合物的结构进行了分析鉴定。7个化合物中有两个是酚性的葡萄糖苷类成分：西藏胡黄连酚苷D (1)、4-O-β-D-(6-O-vanilloyl glucopyranosyl) vanillic acid (6)；四个苯乙基苷类化合物：plantamajoside (2)、plantainoside D (3)、西藏胡黄连苷A (4) 和西藏胡黄连苷F (5)；一个苯基小分子化合物：香豆酸甲酯 (7)。其中化合物1和5未见文献报道，确定为新化合物；化合物3为首次从该种植物中分到。 第二章为鸡矢藤化学成分研究。从鸡矢藤（Paederia scandense (Lour) Merrill）全草中分离出25个化合物，通过理化常数和波谱数据鉴定了它们的结构。25个化合物中包括一个蒽醌类成分：茜根定-1-甲醚 (1)；两个香豆素：异东莨菪香豆素 (2)和5-羟基-8-甲氧基吡喃香豆素 (3)；两个香豆素-木脂素化合物：臭矢菜素 B (4)和臭矢菜素 D (5)；一个木脂素：异落叶松树脂醇 (6)；两个黄酮：diadzein (7)和蒙花苷 (8)；三个三萜类化合物：齐墩果酸 (9)、乌苏酸 (10)和 3-O-β-D-吡喃葡萄糖基乌苏烷 (11)；三个甾体及其糖苷：b-谷甾醇 (12)、胡萝卜苷 (13)和(24R)-豆甾-4-烯-3-酮 (14)；六个小分子化合物：对羟基苯甲酸 (15)，咖啡酸 (16)，香豆酸 (17)，丁烯二酸 (18)，3,5-二甲氧基-4-羟基苯甲酸(19)，咖啡酸-4-O-β-D-吡喃葡萄糖苷(20)；五个环烯醚萜类化合物：鸡矢藤苷 (21)，鸡矢藤酸 (22)，鸡矢藤酸甲酯 (23)，saprosmoside E (24)和paederoside B (25)。其中化合物25未见文献报道，为新化合物。化合物1～8、11、14、15～20为首次从该化合物中分离得到。同时对鸡矢藤中环烯醚萜类化合物做了高效液相-串联质谱(HPLC-MSn)分析，探讨了这类化合物的质谱裂解规律。 第三章为黄芪多糖的提取工艺研究。首先确定了黄芪多糖含量的测定方法，并进行了方法学验证；其次探讨了黄芪中黄芪多糖的提取工艺，确定以酶法-Sevag法联用来去除黄芪多糖中的蛋白质，可使其提取物中黄芪多糖总含量达到70％以上。 第四章为近年来植物多糖的研究进展。主要包括植物多糖的提取纯化、多糖的定性定量检测方法、多糖的结构分析和多糖的药理活性。 This dissertation consists of four parts. The first and second parts reports the studies on the chemical constituents of medicinal plants of Picrorhiza Scrophulariiflora and Paederia scandens. The third part is about the extract technique of Astragalan Polysaccharide (APS). The last part reviews the progress of the studies on plant polysaccharides. 　 The first chapter is about the chemical constituents of P. Scrophulariiflora which is widely used as an important medicine to treat various immune-related diseases. A new phenyl glycoside, scrophenoside D (1) and a new phenylethyl glycoside, scroside F (5), together with five known compounds, plantamajoside (2), plantainoside D (3), scroside A (4), 4-O-β-D-(6-O-vanilloylglucopyranosyl) vanillic acid (6); and methyl-p-coumarate (7) were isolated from the stems of P. scrophulariiflora. Their structures were elucidated by spectroscopic and chemical methods. The second chapter is about the chemical constituents of medicinal herb of P. scandens. Twenty-five compounds were isolated and purified by normal and reversed phase silica gel column chromatography. By physicochemical properties and spectral analysis, their structures were identified as rubiadin-1-methylether (1), isoscopoletin (2), 5-hydroxyl-8-methoxyl-coumarin (3), cleomiscosin B (4), cleomiscosin D (5), isolariciresinol (6), diadzein (7), linarin (8), oleanolic acid (9), ursolic acid (10), 3-O-β-D-glucopyranosyloxyl-ursane (11), b-sitosterol (12), b-daucosterol (13), (24R)-stigmast-4-ene-3-one (14), p-hydroxyl-benzoic acid (15), caffic acid (16), coumaric acid (17), trans-butenedioic acid (18), 3,5-dimethoxyl-4-hydroxylbenzoic acid (19), caffeic acid 4-O-β-D-glucopyranoside (20), paederoside (21), paederosidic acid (22), paederosidic acid methyl ester (23), saprosmoside E (24), paederoside B (25). Among them, compound 25 is a new compound. Compounds 1～8、11、14、15～20 were isolated from this plant for the first time. Futhermore, we studied the HPLC-MSn analysis and investigation of fragmentation behavior of the sulfur-containing iridoid glucosides. The third chapter is about the extracting process of Astragalan Polysaccharide (APS). The method of the content determination is built. The optimum condition of extraction of polysaccharides from Radix Astragali is defined and the more effective way to remove protein is combined enzyme method with Sevag method, by which the content of polysaccharides extract can be up to 70%. The last part is a review of the research progress of the plant polysaccharides, which includes its extraction, isolation, purification, determination, structure analysis, and pharmacology.

自养氨氧化混合种群特性和共存机理初步研究

组特殊自养氨氧化混合种群，表现：无机环境种群生长迅速、生物量高；在一个完全无机的自养生长环境中，不仅保持高氨氧化速率，并出现丰富的异养微生物种群；该种群置于异养、厌氧环境中，迅速表现出产氢特征。对于这样一个特殊的生态体系，研究其共生机理，以及联接这些种群之间的碳源和能源问题，将具有非常重要意义。我们拟从种群特征、细胞表面分泌产物、游离体系产物多糖、蛋白和脂肪酸方面开展研究。 第一部分，自养氨氧化混合种群的基本特征。采用氨氧化培养基，进行种群氨氧化特征研究；采用扫描电镜观察自养混合种群的微观特征；沉降、离心去除微生物种群，分析水相中的总有机碳、糖类等物质；利用LB培养基进行种群的分离、纯化，并采用DGGE手段对微生物种群结构进行分析。结果表明，接入菌种后（2/5000（V/V）），培养液中氨（200mg/L）在3－5天内快速降解；亚硝酸盐与氨氮变化呈负相关趋势，仅有少量硝酸盐含量（< 30mg/L）。氨氧化种群的生物量增长与氨氧化趋势一致，初始生物量7.75 mg/L(蛋白含量)，3-5天后生物量快速增长，并达到最高63.06 mg/L（蛋白含量）。电镜图片显示，种群外包裹一层粘液。离心除去菌体后，检测培养液总有机碳和糖的含量，同样表现出与生物量增长相似的特征，分别由初始的3.73、2.35 mg/L，3－5内天迅速增加，并分别达到最大值35.19、27.45 mg/L。经初步分离、纯化并对纯化菌株进行测序，获得了10株异养微生物分别为布鲁氏菌科苍白杆菌属、纤维单孢菌、类芽孢菌属、黄杆菌属、无色杆菌、鞘脂单胞菌、嗜麦芽寡养单胞菌、噬氢菌属、硫红球菌、假单胞菌；DGGE显示，约有20分条离带，我们对其中的两条优势条带进行切割回收测序，鉴定为欧洲亚硝化单胞菌（Nitrosomonas eur）。 第二部分：混合种群自养-异养菌共生的可能机制。在对微生物种群特征初步分析基础上，针对胞外糖类组分可能被微生物代谢分解，我们重点对微生物细胞蛋白质与糖类进行分析。采用超声结合RIPA裂解液裂解，SDS-PAGE电泳分析混合种群总蛋白种类，并通过氨基酸分析仪及红外光谱法分析氨基酸组成及蛋白红外特征。采用超声破碎结合反复冻融对细胞样品进行处理，提取液采用醇沉、Sevage脱氮白，凝胶过滤方法脱盐和分级分离。对提取物的糖分析包括：紫外扫描，红外光谱，核磁共振，单糖组成分析；扫描电镜观察菌群破裂现象。SDS-PAGE分析结果表明：氨氧化种群不同生长阶段都显示出42kD蛋白表达量很高，d4时42kD蛋白表达已经很强，4-7d内一直持续这种过量表达，直到d8后表达开始减弱。说明42kD蛋白可能与氨氧化密切相关。红外光谱分析显示：细胞提取物的特征峰分布在3427.42cm-1、1718.18 cm-1和1681.72 cm-1、1160.07和1086.74 cm-1，分别对应为OH、 C=O、C－O－C基团，表明具有蛋白的典型特征；氨基酸分析显示蛋白中的Gly,Asp,Ala,Glu含量相对较高。 提取物中胞外多糖分离谱图得到不均一组分，共得到6个收集峰；紫外扫描在201－213 nm处有多糖吸收峰，同样表明多糖成分不均一性；多糖红外光谱特征峰主要分别在3400.49 cm-1、2920.28 cm-1、1154.54和1087.52 cm-1，对应OH、－CH2－ or CH 、C－O－H or C－O－C等多糖特征基团；多糖提取物核磁共振1H d4.3～5.9之间出现强吸收峰，这是1H中，多糖存在的明显证据，1H NMR中，其中O-乙酰基的甲基上的氢信号为d1.1～1.3之间。糖肟全苯甲酸酯衍生物的HPLC测定中，得到单一的单糖峰，由于时间问题，还未进行更深入的试验；电镜图片显示，种群中的细胞有大量的破裂现象。 实验表明，自养氨氧化混合种群显示出快速的氨氧化速率，氨氧化过程生物量和有机质的增加明显。微生物种群包裹粘液层，并分离纯化出大量的异养菌；去除菌体后的游离培养液中存在有机质（包括多糖）说明无机自养生长体系中存在异养菌生长、繁殖的二次碳源；细胞提取物中蛋白条带数目多、种类丰富；细胞多糖提取物具有明显的多糖特征，以及单糖的存在。结合种群的显微特征和游离体系中的有机质的检测结果，我们认为，无机自养生长体系中，种群细胞生长过程中发生的破裂现象可能是导致大量的蛋白、多糖释放到游离胞外，并成为其他异养菌生长的碳源和氮源。这可能是自养体系中，大量异养菌共生的可能机制，至于是什么原因引起种群生长过程中产生的破裂现象，还有待下一步深入研究。 A group of mixed autotrophic ammonia oxidizing populations, having much biological characteristic tested by concerned personnel for pilot test: Performed rapid population growth and obtained high biomass in inorganic environment; Not only maintained a high rate of ammoxidation, promoted a wealth of heterotrophic microbial populations growth in a totally inorganic and autotrophic growth environment; Placed in heterotrophic and anaerobic environment,had the performance characteristics that could rapidly produce hydrogen.For such a special ecological system, Study its symbiotic mechanism and the connection between these populations of carbon and energy issues, will have a very important significance. We intended from the characteristics of the population, the secretion product of cell surface, free substance in the liquid medium like polysaccharide, protein and fatty acids carrying out research. Part I: The basic features of mixed autotrophic ammonia oxidizing populations . Use inorganic liquid medium, processed study for ammonia oxidation characteristics of the population; we used scanning electron microscopy to get micro-features of autotrophic ammonia oxidizing populations .The medium was carried out settlement and centrifugal then removed the microbial populations, after all of that we analysis the water phase for total organic carbon(TOC), carbohydrate and other substances; Solid ammonia oxidizing medium was adopted to separation and purification of population, DGGE means was for structure analysis of microbial population. The results showed that after the inoculum of bacteria (2 / 5000 (V / V)), ammonia in the culture medium (200 mg / L) was rapid degradation in 3-5 days; ammonia and nitrite have the negative correlation between changes in the trend, then only a small amount of nitrate content (<30mg / L). The biomass growth of ammoxidation population in line with the trend of ammonia oxidation, the initial volume of it was 7.75 mg / L (protein content), in 3-5 days upto 63.06 mg / L (protein content). Electron microscope image showed, the populations were wrapped in a layer of mucus, including the a large number ruptted micorbe , Centrifuge to remove bacteria, then detected the medium for total organic carbon and sugar content, result took on the same characteristics with biomass growth, that were from the initial 3.73、2.35 mg / L respectively, in 3-6 days achieved rapid increase in the maximum to 35.19、27.45 mg / L respectively. After initial separation、 purification ,then processed sequencing to strains purified and got the result that there were 10 heterotrophic microorganisms : Brucella Branch pale bacillus, Cellu lomonas, Bacillus species category, a Flavobacterium, colorless Bacteria, Aeromonas sheath fat, little support maltophilia Aeromonas, macrophages species hydrogen, sulphur-MI, Pseudomonas bacteria spores; DGGE display, there were 20 separation bands approximately. Part II: Mixed populations that autotrophic - heterotrophic bacteria symbiotic mechanism. On the basis of preliminary analysis of microbial population characteristics, aiming at extracellular carbohydrate components might be decomposition by microbial, we focused on microbial cell protein and carbohydrate analysis. Using ultrasound combined with RIPA lysis cracking the cells, SDS-PAGE electrophoresis analysis the total protein species of the population, and through the amino acid analyzer studied the compositions of amino acid and infrared spectroscopy analysis of a protein infrared characteristics. Using ultrasound combined with repeatedly freezing and thawing to treated the cell sample, then took the means that alcohol precipitation, deproteinization by Sevage, gel filtration aimed at desalination and grade separation to deal with the lysates . The extraction of sugar analysis included: UV scanning, IR, NMR, single-sugar composition analysis. SDS-PAGE analysis showed that: 42 kD protein expression was very high at different growth stages of mixed autotrophic ammonia oxidizing populations , on the fourth day, 42 kD protein expression had been very strong, 4-7d, it had continued this excessive expression, then started to weaken after 7 days. 42 kD protein that might be closely associated with ammonia oxidation. Infrared spectral analysis showed that: cell extracts with the characteristic that the peak distribution in 3427.42 cm-1、1718.18 cm-1 and 1681.72 cm-1、1160.07 cm-1 and 1086.74 cm-1 corresponding to OH、C = O、C-O-C Groups which had the typical characteristics of protein; and analysis showed that amino acids including Gly, Asp, Ala, Glu ,the content in the protein is relatively high. Exopolysaccharide in the extracts had the separation map that it was uneven, received a total of six collection peaks by the detection mode of phenol-sulphruic acid method ; ultraviolet scan in the 201-213 nm department had polysaccharide absorbing peak, the same ingredients that polysaccharide heterogeneity; infrared polysaccharide spectral characteristics of the main peak at 3400.49 cm-1, 2920.28 cm-1, 1154.54 and 1087.52 cm-1, corresponding OH,-CH2-or CH, C-O-H or C-O-C;and other characteristics of polysaccharide group; 1H NMR of polysaccharide extract appeared absorption peak between d4.3 ~5.9, which is the apparent evidence of polysaccharide, In 1H NMR, the hydrogen signal of one of O-acetyl was between 1.1 to 1.3. The determination of Sugar oxime whole benzoate derivatives by HPLC, there was a single-sugar peak, as a matter of time, yet more in-depth test. Summary: Mixed autotrophic ammonia oxidizing populations show us that it had the ability in ammonia oxidizing and it was great, organic matter and biomass increased significantly in the process of ammonia oxidation. Microbial populations was wrapped up slime layer, the phenomenon of cell breakdown obviously, and there were a lot of separation and purification of the heterotrophic bacteria; a lot of organic matter (including polysaccharides)remined in the medium that removal of cell indicated the inorganic system existed secondary carbon sources that could be used by the heterotrophic bacteria ; there were a large number proteins bands of cell extract, rich variety; cell extracts of polysaccharide had obvious characteristics of polysaccharide, and the existence evidence of single-sugar. Combined population of microscopic characteristics and free of organic matter in the test results, we believe that the health of inorganic system, population growth occurred in the course of the breakdown of the phenomenon is likely to lead to a lot of protein and polysaccharide released into the extracellular free, And other heterotrophic bacteria use them to the growth as carbon and nitrogen. This may be autotrophic system, the large number of heterotrophic bacteria symbiotic mechanism.

川牛膝多糖的免疫活性及其水解活性片段分离研究

川牛膝多糖（CP）是从传统中药川牛膝（Cyathula officinalis Kuan）中提取的一种活性多糖，现代药理研究表明川牛膝多糖是川牛膝许多生物活性的物质基础。本实验室前期进行了川牛膝多糖的提取、分离、结构鉴定及其部分活性研究，发现川牛膝中多糖含量非常高，在对川牛膝多糖活性的初步研究中也证实了其具有免疫调节作用。我们为了进一步了解其免疫调节活性，并为构效关系的研究奠定基础，对其进行了如下研究： 1． 通过体外毒性检测、淋巴细胞增殖实验、NK细胞杀伤活性和腹腔巨噬细胞吞噬中性红活性测定，发现川牛膝多糖在10～300μg/mL浓度范围内，对细胞无毒性作用；能够促进LPS诱导的B淋巴细胞增殖(P<0.01)、增强NK细胞杀伤活性(P<0.05)和PMΦ吞噬中性红活性(P<0.01)，且随多糖浓度增高而增强；但其对ConA诱导的T淋巴细胞的增殖无促进作用(P>0.05)。 2． 通过正常小鼠体内淋巴细胞转化实验、迟发型变态反应分析、抗体生成细胞检测、碳粒廓清检测、腹腔巨噬细胞吞噬鸡红细胞活性和NK细胞活性测定，发现川牛膝多糖在适应性免疫方面能够促进SRBC免疫小鼠体内的抗体生成细胞的生成（P<0.01）和增强DNFB诱导的DTH（P<0.05），但对ConA诱导的脾淋巴细胞增殖无促进作用（P>0.05）；在固有免疫方面能够提高小鼠碳粒廓清速率（P<0.05），PMΦ吞噬 CRBC 活性（P<0.01）和NK细胞杀伤活性（P<0.05）。同时还发现其对由环磷酰胺(Cy)引起的白细胞数下降具有很好的抑制作用（P<0.01）。 3． 为了获得结构明确、均一的保留活性的川牛膝多糖片段，为其作用机制、构效关系研究提供关键研究材料，我们开展了“保留免疫活性的最小片段”的分离制备的初步研究。建立并优化了川牛膝多糖的酸水解条件，发现在6%的样品浓度，0.025mol/L的硫酸浓度，65℃的水解温度，水解时间为8min的条件下可以得到一系列连续的多糖片段；采用Bio-Gel P2 分子筛柱层析分离得到5个级分，通过体外淋巴细胞增殖实验、NK细胞活性测定、腹腔巨噬细胞吞噬中性红实验发现其中的一个片段仍保留较强的免疫活性，并测得其分子量约为2057Da，为保留免疫活性的最小片段的进一步分离奠定了基础。 Cyathula officinalis Kuan is a commonly-used Traditional Chinese Medicine (TCM) with a wide range of pharmacological activities. Modern pharmacological researches showed the polysaccharide extracted from it (CP) is an important component for many bioactivities of this TCM. In the previous studies, we found CP showed significant immuno-regulative activities. In order to evaluate this activity systematically and lay foundations for revealling its immuno-regulative machanisms and the Structure -Function relationship, we carried out the following research works: 1． The in vitro immunoactivities of CP were evaluated by using normal mice immunocytes with respects to cytotoxicity, lymphocytes proliferation, NK activity and the ability of peritoneal macrophage phagocytizing neutral red. The polysaccharide showed no cytotoxicity below the concentration of 300 μg/mL, and could promote B lymphocytes proliferation (P<0.01), enhance NK activity (P<0.05) and the ability of peritoneal macrophage phagocytizing neutral red (P<0.01) at the concentration of 10-300 μg/mL. The above effects were positively correlated with the concentration of the polysaccharides. But it could not promote T lymphocytes proliferation (P>0.05). 2． The in vivo immunoactivities of CP were observed on normal mice through the following indices: splenic lymphocyte transformation efficiency, delayed-type allergy, antibody-forming cells activity (AFC), rate of carbon clearance, rate of peritoneal macrophage phagocytizing chicken red blood cell (CRBC) and NK activity, and its influence on the decline of the mouse leucocyte count induced by Cy. The polysaccharide at medium-dose enhanced delayed-type allergy （P<0.05）and NK activity（P<0.05） and increased the rate of carbon clearance（P<0.05）, AFC activity（P<0.01） and the rate of peritoneal macrophage phagocytizing CRBC（P<0.01）. The polysaccharides also effectively resisted the decline of the mouse leucocyte count induced by Cy（P<0.01）. However, it couldn’t increase the splenic lymphocyte transformation efficiency（P>0.05）. 3． Attempting to isolate and prepare the minimal fragments retaining activity with identical structure for further studying on immuno-regulative mechanism and Structure-Function relationship, we carried out the study on hydrolysis of CP, isolation of hydrolysed fragments, and the activity evaluation of the isolated fragments. CP with concentration of 6% was hydrolysed at 65℃ for 8 min with sulfuric acid of 0.025 mol/L，then the hydrolysate was separated using Bio-Gel P2 chromatography, 5 portions of fragments were obtained. The immunoactivities of these fragments were evaluated by using normal mice immunocytes with respect to lymphocytes proliferation, NK activity and ability of peritoneal macrophage phagocytizing neutral red. One fragment with relative molecular mass of 2057Da was found retaining immunoactivity.