海带多糖清除氧自由基的活性及机理

通过比较不同分子量和化学组成的海带硫酸多糖和褐藻胶的清除氧自由基的活性,探讨了海带多糖清除氧自由基活性的构效关系和作用机理。研究发现,分子量为6~15 ku、糖醛酸含量20.4%的低硫组分F-A2清除自由基活性羟基自由基和超氧阴离子自由基活性高于大分子量的组分F-A和F-B,而高硫低分子量组分清除氧自由基的活性非常低。酶降解得到的低分子量褐藻胶组分清除自由基活性随分子量的降低而升高,明显高于其他硫酸多糖,说明硫酸根对低分子量糖与自由基的反应有阻碍,而多糖中糖醛酸含量越高,清除自由基活性越好。大分子量的海带硫酸多糖经铜离子和H₂O₂反应产生的羟基自由基氧化降解以后,直接可以得到两个比较集中的低分子量岩藻聚糖硫酸酯Fa2(分子量为7 ku)和Fa1(分子量为1 ku),其化学组成和清除自由基活性的比较说明自由基首先降解糖醛酸含量高的硫酸糖片断,甘露糖、半乳糖和葡萄糖形成的糖苷键很容易被自由基氧化降解,而高硫高岩藻糖部分不易被自由基水解,研究结果说明,海带多糖的抗氧化活性不仅与分子量和硫酸根含量有关,糖醛酸、岩藻糖含量和糖链上中性糖的组成对多糖的清除自由基活性都有影响。     

紫菜活性肽复合酶法制备与清除羟自由基作用

以条斑紫菜为原料,利用胰蛋白酶和木瓜蛋白酶组成的复合酶水解紫菜蛋白制备生物活性肽并研究清除羟自由基作用。采用单因素试验考察胰蛋白酶与木瓜蛋白酶的比例、底物质量浓度、酶用量、水解温度、水解时间、pH对羟自由基清除率及水解度的影响,响应面法优化酶解条件,SephadexG-15凝胶层析法测定活性肽的分子质量分布。试验结果表明,复合酶最佳酶解工艺条件为胰蛋白酶与木瓜蛋白酶复合酶比例1.67,底物质量浓度20mg/mL,酶用量1.67%,pH 7.0,温度55℃,酶解4h,紫菜活性肽对羟自由基的清除率为80.6%,清除率为50%时的质量浓度为0.744mg/mL,分子质量大多分布于500～1500ku。     

大珠母贝（Pinctada maxima）金属硫蛋白cDNA克隆与序列特征分析

金属硫蛋白（Metallothionein,MT）是一类普遍存在于生物体内、分子量低、半胱氨酸含量丰富、能够与二价重金属离子结合的蛋白质。MT在清除自由基、解除重金属毒性、参与体内微量元素代谢、防止细胞癌变等方面具有重要作用。本研究通过构建大珠母贝外套膜全长cDNA文库,首次克隆得到大珠母贝金属硫蛋白（PmMT）全长cDNA序列。该序列全长599bp,5’UTR（Untranslated Region）为75bp,3’UTR为296bp,开放阅读框（Open Reading Frame,ORF）为228bp,编码75个氨基酸。编码的氨基酸中半胱氨酸含量丰富,达到29．3％;赖氨酸和甘氨酸含量也较高,均为9．3％;不含苯丙氨酸、组氨酸、色氨酸、酪氨酸等芳香族氨基酸;含有软体动物等无脊椎动物金属硫蛋白的特征序列。序列特征分析表明,该序列具备金属硫蛋白的典型特征,是金属硫蛋白家族成员。     

海参水煮液酶解多肽的抗氧化活性

以海参水煮液的冻干粉为原料,选取中性蛋白酶、胃蛋白酶和胰蛋白酶分别酶解制备多肽,测定其对·OH、O2-·和DPPH·自由基的清除能力,并与合成抗氧化剂TBHQ对比.结果表明,3种蛋白酶酶解制得的海参水煮液多肽对·OH、O2-·和DPPH·自由基的清除能力均随样品质量浓度的升高而增强;其中,中性蛋白酶酶解制得的海参水煮液多肽清除·OH、O2-·和DPPH·自由基的IC50值依次为8.565、6.658和2.015mg/ml,其对O2·的清除能力优于TBHQ.经液相色谱法测定,中性蛋白酶、胃蛋白酶和胰蛋白酶酶解制得的海参水煮液多肽分子量分别分布在＜2 500、＜3 500和＜1 000 D的小分子肽中.     

低分子量海带岩藻聚糖硫酸酯的清除活性氧自由基和体内抗氧化作用

用化学发光分析法研究了3种低分子量涨带岩藻聚糖硫酸酯（LMWF－M，LMWF－I，LMWF－IV）体外清除超氧阴离子自基基（O2^-)及羟基自同基（．OH）的作用，并观察了LMWF－M对高脂血症大鼠的抗氧化作用，结果表明阴离子分量海带岩藻聚糖硫酸酯组分均有清除活性氧自由基的能力，随体系中LMWF浓度的增加，其清除活氧自由基的能力增强，LMWF－I清除自同基的能力最强，它对O2^-的IC50为0.044mg.mL^-1,对.OH的IC50为0.062mg.mL^-1,，LWMF－M能显著降低高脂血症大鼠箅清和组织中LPO含量（P＜0．01），增强SOD活力（P＜0．01）.     

海芦笋黄酮的抗氧化作用及对CCl4致小鼠急性肝损伤的保护作用

为深入探究海芦笋黄酮的活性作用,研究海芦笋黄酮在体外的抗氧化作用及其对CCl_4导致的小鼠急性肝损伤的预防保护作用。通过分析海芦笋黄酮对4种自由基(DPPH自由基、ABTS自由基、羟基自由基及超氧阴离子自由基)的清除能力反映海芦笋黄酮的体外抗氧化能力。将海芦笋黄酮分为低、中、高3个剂量[75、150、300 mg/(kg·d)]对小鼠连续灌胃8 d,通过腹腔注射CCl_4建立小鼠急性肝损伤模型,测定海芦笋黄酮对血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、碱性磷酸酶(ALP)的活性以及肝脏组织中超氧化物歧化酶(SOD)、丙二醛(MDA)、过氧化氢酶(CAT)、谷胱甘肽(GSH)水平的影响,观察肝脏病理变化。结果显示,在体外抗氧化实验中,海芦笋黄酮对4种自由基都有良好的清除效果。在CCl_4急性肝损伤模型中,与模型组对比,各剂量海芦笋黄酮均能使血清中ALT、AST、ALP酶活性、肝脏MDA含量显著降低;使肝脏SOD活性、GSH含量显著增加。海芦笋黄酮中、高剂量组与模型组相比,肝脏CAT活性显著升高。肝脏切片结果显示各剂量组肝组织损伤情况有不同程度改善。海芦笋黄酮对由CCl_4引起的小鼠急性肝损伤具有一定程度的预防保护作用,其急性肝损伤保护机制可能与黄酮对脂质过氧化程度的削弱、机体抗氧化能力的提高有关。     

Oxidative status and histological changes in sea bass larvae muscle in response to high dietary content of docosahexaenoic acid DHA

In previous studies, we observed dystrophic alterations in muscle of 48‐day‐old sea bass fed imbalanced docosahexaenoic acid (DHA) and vitamin E diets. To understand the whole pathological process associated with oxidative stress, a histological study was performed by feeding 14‐day‐old sea bass larvae with microdiets containing different ratios of DHA/vitamin E (1/150, 5/150 and 5/300) for a period of 21 days. Larvae fed diet 1/150 showed no lesions in contrast to larvae fed diets 5/150 and 5/300 where the highest incidence of muscle lesions and thiobarbituric acid reactive substances (TBARS) content was observed. Semithin sections revealed focal lesions consisting of degenerated fibres with hypercontracted myofilaments and extensive sarcoplasm vacuolization affecting both red and white muscle. Ultrathin sections of degenerating muscle fibres showed diffuse dilatation of sarcoplasmic reticulum, disorganized myofilaments and autophagic vacuoles containing myelin figures and dense bodies. Additionally, some macrophages were observed among injured fibres as numerous satellite cells. Results from the study agree with those obtained from previous work, proving the pathological potential of free radicals in sea bass larvae musculature. Moreover, high vitamin E inclusion could not completely protect cell membranes from free radicals action.     

可溶性南极磷虾蛋白-芦丁复合物的制备及抗氧化性能分析

为了提升可溶性南极磷虾蛋白的抗氧化性能,将芦丁复合到可溶性南极磷虾蛋白(SAKP)上,成功制备了SAKP-芦丁复合物,一种新型的多酚-蛋白复合物.通过测定SAKP-芦丁复合物清除二苯基苦基苯肼自由基(DPPH?)和羟基自由基(OH?)的能力来评估其抗氧化能力.同时采用H2O2诱导HepG-2细胞氧化损伤模型评价了SAK...     

舌状蜈蚣藻蛋白质的提取及其抗氧化活性研究

该研究以舌状蜈蚣藻(Grateloupia livida)为原料,以蛋白质提取率为指标,探究不同破壁技术对舌状蜈蚣藻蛋白质的提取效果,并通过单因素分析和正交试验对超声波辅助水提法进行工艺优化,所得蛋白质经硫酸铵盐析法粗提纯后考察其体外抗氧化能力。结果显示,溶胀法、反复冻融法、珠磨法的最高蛋白质提取率分别为(29.66±0.86)%、(24.52±0.04)%、(26.52±0.79)%,均低于超声波辅助水提法;超声波辅助水提法提取舌状蜈蚣藻蛋白质的最佳工艺为:液料比160 mL·g^?1,超声全程时间60 min,超声功率1440 W,pH 6.0,此条件下蛋白质提取率可达58.16%;舌状蜈蚣藻粗蛋白质量浓度在1~8 mg·mL^?1内均具有较强抗氧化活性,其质量浓度为8 mg·mL^?1时的还原力为0.43±0.01,对1,1-二苯基-2-三硝基苯肼(DPPH)自由基的半数清除浓度(IC50)为4.00 mg·mL^?1,对2,2'-二氮-双(3-乙基苯并噻唑-6-磺酸)(ABTS)自由基的IC50为3.96 mg·mL^?1。     

Peroxidase activity as a measure of neutrophil populations in inflammatory peritoneal exudates of rainbow trout, Oncorhynchus mykiss (Walbaum)

A quantitative peroxidase assay using tetramethylbenzidine was applied to exudates from peritoneal cavities of rainbow trout which had been inflamed by intraperitoneal injection of Yersinia ruckeri . Most of the peroxidase activity was cell-associated, with values for extracellular peroxidase below 5% of total peroxidase. No free peroxidase activity was detected in resting cavities. The kinetics of neutrophil numbers and peroxidase activity in peritoneal exudates during the 6 days of inflammation studied followed identical patterns, with a correlation coefficient of 0.996. This shows that the peroxidase assay can be used as an indirect, quantitative evaluation of neutrophil responses during peritoneal inflammation.     

缢蛏多糖的提取及抗氧化作用研究

以新鲜缢蛏的碎肉为材料,加水配制成一定比例的匀浆,利用超声波提取法对缢蛏碎肉匀浆进行多糖的提取。利用蒽酮—硫酸法对多糖得率进行计算。将得到的多糖粗品通过DEAE离子交换层析及G-75凝胶层析进一步分离纯化。通过邻苯三酚自氧化体系和Fenton体系测定所得样品的抗氧化能力。结果表明:利用超声波提取多糖的提取率为19.93%。多糖粗品通过DEAE离子交换柱,得到2个峰,峰1对超氧阴离子的清除能率为43.89%,质量浓度为1 mg/ml时,对.OH清除率为57.80%。峰2对超氧阴离子的清除能率为57.80%,质量浓度为1 mg/ml时,对.OH清除率为77.83%。将DEAE峰2进行G-75凝胶层析得到2个峰,峰1对超氧阴离子的清除能率为83.08%,质量浓度为1 mg/ml时,对.OH清除率为94.76%。峰2对超氧阴离子的清除能率为74.41%,质量浓度为1 mg/ml时,对.OH清除率为52.41%。     

茶多酚的生物学功能及在畜牧业中的应用概况

茶多酚具有清除自由基、抗氧化等生物学功能,在杀菌、抗病毒、抑制肿瘤、防癌抗癌、控制心血管疾病、免疫调节等方面具有良好功效。在抗生素、生长激素的毒副作用日益遭到质疑的今天,茶多酚以其生物学功能和“绿色”本质倍受关注。文章就茶多酚的组成、结构、生物学功能、作用机制及其在畜牧业中的应用展开综述。     

Antioxidant Activity of Hydrolysates and Peptide Fractions of Nemipterus japonicus and Exocoetus volitans Muscle

The focus of the study was to investigate the antioxidant activity of hydrolyzed muscle protein of Nemipterus japonicus and Exocoetus volitans. The trypsin protein hydrolysates of both fish showed maximum free radical scavenging potential and lipid peroxidation inhibition. Furthermore, it was purified by chromatographic methods followed by the lipid peroxidation inhibition; free radical scavenging assay was performed before and after purification. The purified peptide fractions of N. japonicus and E. volitans exhibited higher activity against polyunsaturated fatty acids (PUFA) peroxidation which was similar to natural antioxidants like α-tocopherol. Free radical scavenging potencies were measured by electron spin resonance technique (ESR). The purified peptide of E. volitans quenched free radicals (DPPH, hydroxyl, and superoxide) slightly more than N. japonicus. The amino acid composition of both fish protein hydrolysates showed variations in their ratio. The purified peptides were tested for cell cytotoxicity for Vero (kidney epithelial cells of the African Green Monkey) and Hep G₂ (human hepatocellular liver carcinoma) cell lines. It was found that peptides did not show any cytotoxic effect for Vero cell lines and exerted a significant antiproliferative effect on Hep G₂ cell lines.     

Antioxidant and Angiotensin I Converting Enzyme Inhibition Effects and Antihypertensive Effect in Spontaneously Hyertensive Rats of Peptide Isolated from Boiled Abalone By-Products,Hallotis discus hannai

ABSTRACT

Abalone is a marine gastropod that is an important fishery and food industrial resource, massively maricultured in Asia, Africa, Australia, and America. Marine by-products from boiled abalone, normally discarded as industrial waste in manufacturing plants, were fractionated and concentrated using an ultrafiltration membrane system (? 1 kDa). In this study, the antioxidant and angiotensin I converting enzyme (ACE) inhibition effects of the boiled abalone by-products (BABs) were investigated. BABs have high antioxidant activities against free radicals (1,1-diphenyl-2-pycryl-hydrazyl (DPPH), hydroxyl, peroxyl, and superoxide radicals), reactive oxygen species (ROS) stress, and DNA damage in H₂O₂-treated RAW 264.7 macrophages. The structure of the purified peptide was identified to be ATPGDEG (MW 752 Da), and the ACE inhibition pattern of the peptide was found to be noncompetitive. In addition, the peptide exhibited an antihypertensive effect according to time-course measurement after oral administration to spontaneously hypertensive rats (SHR). BABs from this genus may be potential candidates for the development of unique natural substances for further industrial applications as functional foods and pharmaceuticals.     

扇贝内脏团提取物对药物性肝损伤的保护作用

采用生化技术从扇贝内脏团中制备出具有药用价值的扇贝提取物。化学分析结果：牛磺酸含量占１．６％，糖原含量占８．２％，支链氨基酸（ＢＡＣＣ）含量占４０．７１％。支链氨基酸与芳香族氨基酸克分子比（ＢＣＡＡ／ＡＡＡ）为３４．７０。Ｄ—氨基半乳糖肝损伤和四氯化碳肝损伤动物模型试验结果表明，扇贝提取物能提高药物性肝损伤大鼠血清的ＢＣＡＡ／ＡＡＡ和肝组织中抗过氧化酶活性，有纠正血清氨基酸谱紊乱和增强机体抗氧化、清除活性氧自由基能力，对肝脏具有保护作用。     

Influences of Aeromonas salmonicida lipopolysaccharide, prednisolone and water temperature on plasma protein composition in salmonids

To further characterize the putative role of constitutive and inducible plasma proteins in innate resistance to furunculosis, the present authors compared the alterations in profiles of plasma proteins in resistant and susceptible salmonids, i.e. rainbow trout, Oncorhynchus mykiss (Walbaum), and brook trout, Salvelinus fontinalis (Mitchill), respectively. Rainbow trout were injected with prednisolone acetate and exposed to higher water temperature (18 °C versus 10 °C), or injected with purified lipopolysaccharide (LPS) from a virulent strain of Aeromonas salmonicida , and plasma components were examined by two-dimensional polyacrylamide electrophoresis . Two days after A. salmonicida LPS exposure, rainbow trout had a four- to five-fold increase in concentrations of plasma proteins composed of p48, p19 and p16 subunits, and a significant decrease in a 100-kDa protein group. Consistent elevation or depletion of proteins corresponding to previously reported rainbow trout A. salmonicida LPS-binding pentraxins and lectins in plasma were not observed. Brook trout exposed to A. salmonicida LPS did not have any consistent plasma protein changes. There were no significant alterations in major plasma proteins following temperature shock and prednisolone acetate administration in rainbow trout plasma. These studies demonstrate that rainbow trout with LPS-induced sterile inflammation have few alterations in major plasma proteins or LPS-binding proteins, and do not exhibit the spectrum of acute phase changes induced by inflammation in mammals.     

Does dietary tocopherol level affect fatty acid metabolism in fish?

Fish are a rich source of the n-3 polyunsaturated fatty acids (PUFA), particularly the highly unsaturated fatty acids (HUFA) eicosapentaenoic (EPA; 20:5n-3) and docosahexaenoic (DHA; 22:6n-3) acids, which are vital constituents for cell membrane structure and function, but which are also highly susceptible to attack by oxygen and other organic radicals. Resultant damage to PUFA in membrane phospholipids can have serious consequences for cell membrane structure and function, with potential pathological effects on cells and tissues. Physiological antioxidant protection involves both endogenous components, such as free-radical-scavenging enzymes, and exogenous dietary micronutrients including tocopherols and tocotrienols, the vitamin E-type compounds widely regarded as the primary lipid-soluble antioxidants. The antioxidant activities of tocopherols are imparted by their ability to donate their phenolic hydrogen atoms to lipid (fatty acid) free radicals, resulting in the stabilization of the latter and the termination of the lipid peroxidation chain reaction. However, tocopherols can also prevent PUFA peroxidation by acting as quenchers of singlet oxygen. Recent studies on marine fish have shown correlations between dietary and tissue PUFA/tocopherol ratios and incidence of lipid peroxidation, as indicated by the levels of thiobarbituric-acid reactive substances (TBARS) and isoprostanes. These studies also showed that feeding diets containing oxidized oil significantly affected the activities of liver antioxidant defence enzymes and that dietary tocopherol partially attenuated these effects. However, there is evidence that dietary tocopherols can affect fatty acid metabolism in other ways. An increase in membrane PUFA was observed in rats deficient in vitamin E. This was suggested to be due to overproduction of PUFA arising from increased activity of the desaturation/elongation mechanisms responsible for the synthesis of PUFA. Consistent with this, increased desaturation of 18:3n-3 and 20:5n-3 in hepatocytes from salmon fed diets deficient in tocopherol and/or astaxanthin has been observed. Although the mechanism is unclear, tocopherols may influence the biosynthesis of n-3PUFA through alteration of cellular oxidation potential or peroxide tone.     

邻苯二甲酸二丁酯对厚壳贻贝抗氧化防疫系统的影响

邻苯二甲酸二丁酯（dibutyl phthalate,DBP）作为增塑剂,已被中国列入优先控制的污染物名单,其对海洋生物的影响已引起人们的广泛关注。为研究邻苯二甲酸酯类化合物（phthalic acid esters,PAEs）对海洋贝类抗氧化防疫系统的影响,本文以厚壳贻贝（）为受试生物,研究了不同质量浓度DBP（0 mg/L、100 mg/L、200 mg/L、280 mg/L、360 mg/L）胁迫以及清水释放对其鳃和内脏团中抗氧化系统相关指标超氧化物歧化酶（superoxide dismutase,SOD）、过氧化氢酶（catalase,CAT）活性以及丙二醛（malondialdehyde,MDA）含量变化的情况。结果表明：厚壳贻贝鳃和内脏团SOD、CAT活性以及MDA含量呈现不完全相同的变化规律。DBP胁迫阶段,鳃和内脏团中SOD活性基本呈先诱导后抑制再诱导的趋势,CAT活性则先抑制后诱导,MDA含量基本呈不断升高趋势;清水释放阶段,鳃和内脏团中SOD活性基本呈先抑制后诱导的趋势,CAT活性基本处于诱导状态,之后内脏团CAT活性被抑制,MDA含量处于先升高,之后内脏团MDA含量出现降低的趋势,浓度-效应和时间-效应较为明显。研究表明,在DBP胁迫下,厚壳贻贝鳃和内脏团产生了氧化损伤,可诱导机体产生脂质过氧化现象,且15 d清水释放期间不足以让厚壳贻贝恢复到正常水平。本研究旨在为海洋贝类的健康养殖和风险评价提供科学依据。     

Organic components from scallop shell increase expression of keratinocyte growth factor in human skin fibroblast

We observed previously that scallop shells contain organic components that have various useful applications for skin. The organic components from scallop shell (scallop shell extract) enhance the turnover rate of the rat skin epidermal layer and increase the rate of recovery of UVB-induced injury to rat dorsal skin. We investigated the effect of scallop shell extract on the expression of keratinocyte growth factor (KGF) from human skin fibroblasts because it has been reported that KGF promotes wound healing and reduces the generation of UVB-induced free radicals in skin. Scallop shell extract increased the mRNA expression level of KGF in skin fibroblasts and the secretion of KGF from the fibroblasts at the protein level. These results suggest that the scallop shell extract may promote recovery from UVB-induced injury in skin through an increase in the secretion of KGF from the fibroblast cell.     

Melano-macrophage centres and their role in fish pathology

Melano-macrophage centres, also known as macrophage aggregates, are distinctive groupings of pigment-containing cells within the tissues of heterothermic vertebrates. In fish they are normally located in the stroma of the haemopoietic tissue of the spleen and the kidney, although in amphibians and reptiles, and some fish, they are also found in the liver. They may also develop in association with chronic inflammatory lesions elsewhere in the body and during ovarian atresia. In higher teleosts, they often exist as complex discrete centres, containing lymphocytes and macrophages, and may be primitive analogues of the germinal centres of lymph nodes. Melano-macrophage centres usually contain a variety of pigments, including melanins, and these increase in range and volume in older fish or in the presence of cachectic disease. Melano-macrophage centres act as focal depositories for resistant intracellular bacteria, from which chronic infections may develop. Iron capture and storage in haemolytic diseases appears to be a primary function, but antigen trapping and presentation to lymphocytes, sequestration of products of cellular degradation and potentially toxic tissue materials, such as melanins, free radicals and catabolic breakdown products are among other functions that have been ascribed. Recent work suggests that they are a site of primary melanogenesis rather than mere storage. Melano-macrophage centres increase in size or frequency in conditions of environmental stress and have been suggested as reliable biomarkers for water quality in terms of both deoxygenation and iatragenic chemical pollution.