海带磷酸甘露糖变位酶(PMM)基因的克隆与表达分析

磷酸甘露糖变位酶(PMM)是褐藻胶和岩藻聚糖合成过程中的关键酶之一。本研究利用c DNA末端快速克隆(RACE)技术,获得2条海带PMM基因(Sjpmm1,Sjpmm2)序列。其中,Sjpmm1的开放阅读框(ORF)长759 bp,其编码的Sj PMM1为卤酸脱卤酶(HAD)超家族成员,含252个氨基酸,分子量约为28.51 k Da;而Sjpmm2的ORF长1866 bp,其编码的Sj PMM2属于磷酸己糖变位酶超家族的成员,含621个氨基酸,分子量约为66.49 k Da。海带PMM的二级结构均以?-螺旋为主。进化分析表明,Sjpmm1来自于原始真核生物,而Sjpmm2来源于质体的第一次内共生作用。实时定量PCR分析发现,海带受到高温或低温胁迫时,Sjpmm1和Sjpmm2转录水平上升,以合成岩藻聚糖抵抗环境影响。此外,利用p MAL-c5X载体对Sj PMM1进行体外表达,得到高浓度的可溶性融合蛋白,为后续的Sj PMM功能分析提供基础。     

Chemical identification,antioxidant, cholinesterase inhibitory,and cytotoxic properties of fucoidan extracted from Persian Gulf Sargassum angustifolium

Marine macroalgal sulfated fucose-containing polysaccharides, like fucoidan, have drawn significant attention due to their biotechnological potentials, such as anti-cancer, antioxidant, and anti-cholinesterase activities. The fucoidan derived from brown macroalgae Sargassum angustifolium species (FSA) was investigated for its cytotoxic effects and alterations in cell proliferation, and cell cycle-related gene expression in the present study occurred on NB4 cell line. The results showed that FSA would induce p53, p21, pro-apoptotic genes and increase expression of the p15 gene as a cell arrest marker. Also, FSA inhibited the anti-apoptotic effect of the Bcl-2 gene and decreased dnmt-1 gene expression. FSA significantly exhibited potent 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity (p<0.05) with an IC₅₀ value of 0.157 mg/mL and showed moderate anti-acetylcholinesterase activity with an IC₅₀ value of 1.20 μg/mL. These results indicated the potential of FSA for the development of therapeutic or preventive agents of cancer and Alzheimer’s disease mainly through cytotoxic effect and AChE (acetylcholinesterase) inhibition as well as additional antioxidant capacities.     

褐藻多糖硫酸酯对脂多糖诱导大鼠肾小球系膜细胞NO产生量的影响

慢性肾衰的进程中伴随着肾脏炎症的发生,研究表明褐藻多糖硫酸酯具有抗炎作用,且对于慢性肾衰的早期治疗具有一定的作用。本文通过脂多糖(lipopolysaccharide,LPS)诱导,建立大鼠肾小球系膜细胞的炎症模型,用来源于海带的高分子质量褐藻多糖硫酸酯F1和低分子质量褐藻多糖硫酸酯F2对细胞进行处理,24 h后MTT法检测各组细胞活力,Griess法检测24 h及48 h后细胞培养液中NO的含量。结果表明,各组间细胞活力无明显差异;脂多糖可以诱导大鼠肾小球系膜细胞NO产量增加,F1和F2均可在一定程度上抑制脂多糖诱导的NO产量的增加,从而减轻细胞炎症反应。剂量为25μg/m L的F1和F2处理细胞48 h后,与模型组相比,F1和F2组细胞培养液中NO含量分别下降22.52%和38.65%,低分子质量褐藻多糖硫酸酯抗炎效果明显高于高分子质量褐藻多糖硫酸酯。     

褐藻多糖的药理作用与制备

阐述了褐藻糖胶的化学组成和结构。从褐藻中提取分离得到的褐藻糖胶具有显著的抗癌、抗凝、降脂、免疫调节和抗衰老作用,既可作为保健食品,也可作为治疗药物。目前,我国制备褐藻糖胶,主要采用60%乙醇沉淀法和CPC沉淀法从海带水碱凝沉物中提取,或用稀酸直接从海带中提取,并用乙醇分级法和色谱法对褐藻糖胶进行纯化。     

人肠道微生物对海带岩藻聚糖硫酸酯及其寡糖的降解利用

采用人肠道微生物体外厌氧发酵技术,研究人肠道微生物对不同分子量岩藻聚糖硫酸酯的降解利用。分别将五个志愿者的肠道微生物接种到以高分子量和低分子量岩藻聚糖硫酸酯为唯一碳源的培养基中,酵解48h后,采用TLC和PAGE分析分子量变化,PMP-HPLC分析单糖组成变化,GC分析短链脂肪酸的生成情况。结果发现:在人肠道微生物体外降解岩藻聚糖硫酸酯的体系中,岩藻聚糖硫酸酯寡糖和低分子量组分(10—20k Da)含量显著降低,而且酵解产物的单糖组成没有变化,说明被人肠道微生物彻底降解利用;而人肠道微生物对高分子量岩藻聚糖硫酸酯(20k Da)的降解和利用度很低,产物的半乳糖和甘露糖的比例明显下降。低分子量和高分子量岩藻聚糖硫酸酯在酵解后均生成短链脂肪酸乙酸、丙酸和丁酸,但在高分子量岩藻聚糖硫酸酯发酵液中还生成了支链脂肪酸(异丁酸、异戊酸)和戊酸。研究结果表明,人肠道微生物能够彻底降解利用复杂的海带岩藻聚糖硫酸酯寡糖和低分子量组分,但是对高分子量岩藻聚糖硫酸酯的降解率很低。单糖分析说明人肠道微生物能够降解和利用多糖中的所有单糖,产生有利于肠道健康的短链脂肪酸,但是只有低分子量岩藻聚糖硫酸酯能够抑制支链脂肪酸生成。     

海带岩藻聚糖硫酸酯测定方法的研究

应用次甲基蓝分光光度法测定溶液中岩藻聚糖硫酸酯的含量 ,有色化合物的最大吸收波长为 5 6 0 nm ,在 4～ 2 0μg/ m L内线性良好 (r=0 .999) ,回收率为 (96 .2 7± 1.6 6 ) %。在测定过程中 ,无机盐影响较大 ,蛋白质影响很小 ,而 p H值、其它糖没有影响 ,PBS(磷酸盐缓冲液 )能完全消除褐藻胶对测定的影响。实验证明 ,该方法准确、快速、灵敏度高。     

褐藻中岩藻聚糖和岩藻多糖的结构与功能的研究

褐藻中两种硫酸多糖——岩藻多糖和岩藻聚糖含量丰富。它们在结构上相似而不相同 ,都具有抗凝血、抗肿瘤、抗病毒、抑制补体激活、抑制精卵结合、吸收重金属等功能。着重介绍了岩藻聚糖和岩藻多糖在抗凝血、抗肿瘤、抑制补体激活方面的研究进展。     

Analysis of the monosaccharide composition of fucoidan by precolumn derivation HPLC

We developed an HPLC method for analysis of the monosaccharide composition of fucoidans.The fucoidan was hydrolyzed into monosaccharides with 2 mol/L trifluoroacetic acid.Using ribose as the internal standard,the monosaccharide derivatives,obtained with 1-Phenyl-3-methyl-5-pyrazolone(PMP),were separated by reverse-phase HPLC using a gradient elution process,and monitored by ultraviolet detection at 245 nm.In the concentration range of 0.1-2.0 mmol/L,the peak area of each monosaccharide had a good linear rel...     

海带中岩藻聚糖硫酸酯提取工艺评价模型的建立与探讨

采用均匀设计从海带中热水提取岩藻聚糖硫酸酯,以粗多糖提取率(Yw)、岩藻糖提取率(Yf)和硫酸根提取率(Ys)为评价指标,并采用综合加权评分法综合考虑各试验因素对以上三个指标的影响.利用Minitab软件分析加权值Y与各因素的相关性,再通过回归方程优化岩藻聚糖硫酸酯的工艺参数.建立了加权值Y与提取温度(X1)、提取时间(X2)和液料比(X3)之间的二次回归模型,即Y=65.2+0.555x1+1.73x2+0.587x3+0.0236x12+0.0939x32-0.110x1x2.优化后获得最佳提取工艺参数.     

Study on antithrombotic and antiplatelet activities of low molecular weight fucoidan from Laminaria japonica

The antithrombotic and antiplatelet effects of two fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica were compared in order to examine the influence of chemical character on their antithrombotic activity and the possible mechanism. Both LMW fucoidan fractions exhibited favorable antithrombotic activity in an Fecl3-induced arterial thrombosis. The antithrombotic activity of LMW fucoidan was related with decrease of TXB2 and whole blood viscosity and hematocrit. LMW fucoidan showed a correlation between anticoagulant, antiaggregant and antithrombotic effects in vivo. For LMW fucoidan, antithrombotic activity required high dose of 5-10 nmol kg-1, concomitantly with increase in anticoagulant activity and inhibition of platelet aggregation. Administration of LMW fucoidan significantly promoted the 6-keto-PGF1α content and decreased the TXB2 content, indicating its inhibition of tissue factor pathway and regulation of metabolism of arachidonic acid. By comparison, highly sulfated fucoidan LF2 with Mw 3900 seemed to be a more suitable choice for antithrombotic drug for its antithrombotic activity accompanied with specific inhibitory activity on platelet aggregation, low anticoagulant activity and low hemorrhagic risk in vivo.