含N-乙酰化肝素寡糖的制备及序列分析

建立了含N-乙酰化肝素寡糖的分离提纯及其序列结构分析方法.首先应用肝素酶Ⅰ深度酶解低分子量肝素来富集含N-乙酰化结构寡糖,通过Bio-Gel P10凝胶色谱法分离制备了包括二糖至十四糖的系列肝素寡糖粗样品,ProPac PA-1强阴离子高效液相色谱(SAX-HPLC)等方法对粗样品进一步分离,提纯得到4种六糖和3种八糖片段.其次应用肝素酶Ⅰ,Ⅱ和Ⅲ复合酶解与HPLC法分析各纯化寡糖的二糖组分,并结合肝素酶Ⅰ底物特异性,初步推断4种六糖和3种八糖的序列结构.在寡糖的糖链两端均含有N-硫酸化二糖,而N-乙酰化二糖分布在糖链当中.应用电喷雾离子阱-飞行时间质谱(ESI-IT-TOF-MS)在负离子模式下进一步表征寡糖并分析其裂解规律.结果表明,各寡糖中均出现大量因SO32-丢失形成的碎片离子峰,六糖中主要有双电荷和三电荷碎片离子峰;在八糖中出现了一系列从双电荷至五电荷的离子峰.各寡糖的双电荷离子峰质荷比进一步确定了上述寡糖的序列结构.六糖的裂解规律表明,裂解主要存在于糖苷键,N-乙酰葡糖胺和糖醛酸上的裂解方式分别为0,2X和0,2Z.本研究提供了切实有效的分离、分析未知结构肝素寡糖序列的新方法.     

黑木耳寡糖的提取、结构表征及生物活性

以热爆预处理制备的黑木耳发酵原浆为原料, 采用热水浸提法提取黑木耳粗多糖(Crude Auricularia heimuer polysaccharide, CAHP). 通过H₂O₂氧化水解和超声物理水解联用的方法降解CAHP, 经阴离子交换柱层析和凝胶过滤柱层析分离, 获得黑木耳寡糖1(Auricularia heimuer oligosaccharide 1, AHO1). 单糖组成分析、 高分辨质谱、 红外光谱和核磁共振波谱等表征结果表明, AHO1是以甘露糖为主, 由6种单糖组成, 各糖残基通过α-糖苷键连接, 聚合度为2~10的低聚糖. 功能分析结果表明, AHO1具有一定的自由基清除能力, 且能够有效减弱H₂O₂诱导的HepG2细胞氧化应激反应. 抑菌实验结果显示, AHO1不仅对大肠杆菌DH5α和金黄色葡萄球菌ATCC6538具有较好的抑制作用, 还能有效抑制带氨苄抗性的基因工程菌活性.     

人乳寡糖的高分辨质谱结构分析及液相色谱条件的比较

人乳寡糖(Human milk oligosaccharides,HMOs)与婴幼儿生命初期的生长发育密切相关,揭示其组成及结构有助于阐明HMOs功能.本研究通过超高效液相色谱-高分辨质谱联用技术,对19个HMOs(16个中性HMOs、3个酸性HMOs)进行了结构解析,在寡糖异构体裂解规律的基础上详细探讨了4组异构体的...     

κ-卡拉胶寡糖AEC柱前衍生物的LC-ESI-MS/MSn分离分析

以κ-卡拉胶为原料, 经盐酸水解得到一系列寡糖混合物. 以3-氨基-9-乙基咔唑(3-amino-9-ethylcarbazole, AEC)为衍生化试剂, 对酸解得到的κ-卡拉胶寡糖进行柱前衍生, 采用反相C₁₈色谱柱(250 mm×4.6 mm, 5 μm), 乙腈和乙酸铵水溶液(pH 4.5)为流动相, 梯度洗脱, 在254 nm波长处检测, 建立了κ-卡拉胶寡糖衍生物的高效液相色谱(HPLC)分离以及液相色谱-电喷雾质谱联用(LC-ESI-MS)分离分析的方法, 并对AEC衍生后的κ-卡拉胶寡糖进行多级质谱裂解(MSⁿ), 进一步获得了κ-卡拉胶寡糖的结构信息. 该方法对κ-卡拉胶寡糖的结构分析、构效关系等方面的研究有参考价值.     

胶束电动色谱柱上酶反应测定低分子量肝素的抗凝血活性

发展了一种基于胶束电动色谱(MEKC)结合柱上酶微反应的方法,用于测定低分子量肝素(LMWHs)的抗凝血活性。肝素与抗凝血酶Ⅲ(ATⅢ)结合后,将ATⅢ抑制凝血因子10(FXa)活性提高了约1 000倍。通过测定FXa水解生色肽底物CPS产生的对硝基苯胺(p-NP)就可以测定FXa的活性。因此,通过LMWHs抑制FXa产生的对硝基苯胺的量就可以测定抗凝血活性。该方法将毛细管柱端作为微量的酶微反应器,以呋喃妥英(nitrofurantoin, NF)作为内标,依次将LMWHs溶液、ATⅢ溶液、FXa和CPS溶液导入毛细管柱端,反应物经过分子扩散、横向层流扩散混合和电压混合后反应。反应结束后,采用MEKC分离模式将产物对硝基苯胺与底物以及其他大分子分离,在其最大波长380 nm下测定产生对硝基苯胺的量,从而确定LMWHs的抗凝血活性。该方法具有自动化、重复性好、灵敏度高、样品消耗量少的优点,而且不受其他成分的干扰,可用于各种复杂样品(如血浆)中LMWHs抗FXa活性的监测。     

体积排阻色谱法测定低分子量肝素抗凝血因子Xa的活性

发展了一种基于体积排阻色谱测定低分子量肝素(LMWH)抗凝血活性的方法。利用肝素与抗凝血酶Ⅲ(ATⅢ)结合后可增强ATⅢ对凝血因子Xa(FXa)抑制作用的原理,通过测定加入LMWH后FXa水解其生色底物产生对硝基苯胺(pNA)这一反应的抑制程度确定LMWH的活性。首先将含有一定浓度LMWH的缓冲溶液与ATⅢ溶液混合,然后依次加入FXa和生色底物,分别孵育一段时间。底物被FXa水解,产生游离的pNA。体积排阻色谱可将小分子产物pNA与其他大分子分离开,因而可以在pNA的最大吸收波长下得到高灵敏度的测定,并且不再受其他成分的干扰。该方法重复性好,灵敏度高,极大地减少了样品的消耗量,降低了成本,并且还可进行各种复杂样品(如血浆)中LMWH抗FXa活性的监测。     

南天竹花挥发油化学成分的GC-MS分析及体外抗氧化活性

采用水蒸气蒸馏法提取南天竹花挥发油,利用气相色谱-质谱联用技术分析挥发油化学成分;以ABTS自由基清除作用、金属离子螯合作用为指标评价20%挥发油的体外抗氧化活性。从南天竹花挥发油中共鉴定出60种化合物,占挥发油总量的76.98%,主要成分为棕榈酸、棕榈醛、十八烷醛、3,7-二甲基-1,5,7-辛三烯-3-醇、1-十六烯等;挥发油对ABTS自由基和金属离子具有较好地清除作用和螯合作用,且样品量与活性呈量效关系。     

基于电喷雾串联质谱的蓝藻寡糖脱果糖分析及系列α-1,2-葡聚寡糖的制备

为获得系列α-1,2-葡聚寡糖,首先以蓝藻寡糖六糖、八糖、九糖和十糖为原料,在0.5 mol/L的三氟乙酸(TFA)中于95℃酸解9 min以脱去还原端果糖,经低压凝胶色谱分离纯化,用电喷雾离子化-碰撞诱导解离-串联质谱(ESI-CID-MS/MS)和基质辅助激光解吸电离质谱(MALDI-MS)鉴定和序列表征,获得了除去末端果糖的α-1,2-五、七、八和九糖;然后在0.5 mol/L的TFA中于95℃对混合蓝藻寡糖六糖和八糖酸水解45 min,用Bio-Gel P2凝胶柱对混合物进行分离和纯化,并通过ESI-MS和MALDI-MS对获得的每个寡糖组份进行表征,获得了聚合度为2,3,4和6的α-1,2-葡聚寡糖.本研究为利用糖生物芯片技术进行α-1,2-葡聚寡糖的功能筛选及分析其与靶标蛋白之间相互作用的特异性提供了葡聚寡糖物质基础.     

肝素非还原端饱和结构的紫外吸收研究及在肝素寡糖测序中的应用

为了进一步探讨非还原端饱和结构的肝素寡糖在UV 232 nm的吸收情况, 制备了4种饱和结构的肝素二糖, 并用离子对反相液相色谱/离子阱飞行时间质谱(RPIP-LC/MS-IT-TOF)光电二极管阵列检测器分析了它们在UV 232 nm的吸收情况. 分析结果表明, 饱和结构的肝素二糖在UV 232 nm的检出限为9 μg(S/N=10), UV 232 nm/UV 206 nm约为不饱和结构肝素二糖UV 232 nm的7%~40%. 结果还表明, 肝素二糖UV 232 nm的吸收强度受亚硫酸基团(SO₃^2?)影响较大. 另外, 通过比较不饱和结构的肝素/硫酸类肝素(Hep/HS)标样二糖发现, 含N-未取代葡萄糖胺(GlcNH₃⁺)基团的二糖在UV 232 nm的吸收值较低. 最后, 通过简单的UV检测方法, 结合 HNO₂(pH=4.0)裂解法和RPIP-LC/MS-IT-TOF分析, 简化了含GlcNH₃⁺肝素六糖的测序方法. 本研究为以后用 HNO₂(pH=1.5)裂解法对混合组分N-硫酸化的肝素寡糖结构序列分析提供了可能.     

Preparation,Characterization and Anticoagulant Activity of Guar Gum Sulphate

Guar gum was chemically modified by sulphonation using chlorosulphonic acid (ClSO₃H) as a reagent. Effects of molar ratio of ClSO₃H to glucopyranosic unit (ClSO₃H/GU), reaction time and reaction temperature on the degree of sulphonation (DS) and molecular weight (Mw) of products were studied. The structures of guar gum sulphate were investigated by GPC, FT‐IR and UV‐Visible spectroscopy. Activated partial thromboplastin time (APTT) assay showed that the guar gum sulphate could inhibit the intrinsic coagulant pathway. The anticoagulant activity strongly depended on the DS and Mw of polysaccharides. DS>0.56 was essential for anticoagulant activity. The guar gum sulphate with the DS of 0.85 and the Mw of 3.40×10⁴ had the best blood anticoagulant activity.     

肝素杂化材料的制备及抗凝血性质的初步研究

以甲苯二异氰酸酯(TDI)中的-NCO基与纳米金属氧化物表面的羟基发生反应,得到改性纳米金属氧化物,并使其与肝素钠(Heparin)进行接枝反应生成肝素杂化材料,结合红外、热重、扫描电镜(SEM)等表征方法,确定纳米金属氧化物确实接枝到了肝素钠的表面。通过对体外凝血时间和复钙时间的测定,来初步研究肝素杂化材料的抗凝血性质。结果表明：肝素杂化材料的抗凝血时间和复钙时间均比肝素钠的要短,表明它的抗凝血性比肝素钠的抗凝血性要弱一些;但比纳米金属氧化物和空白组的抗凝血时间和复钙时间要长,说明肝素杂化材料的抗凝血性与其相比则有明显的提高。     

Synthesis,Characterization and Antioxidant Activity of Quaternized Carboxymethyl Chitosan Oligosaccharides

In order to determine the effect of quaternary ammonium groups and carboxymethyl groups of chitosan on antioxidant activity, nine quaternized carboxymethyl chitosan oligosaccharides (QCMCOs) were prepared from chitosan with chloroacetic acid and 2,3-epoxypropyltrimethyl ammoniumchloride as the modifying agent under microwave irradiation. The structures of QCMCOs were characterized by FT-IR, NMR, XRD and their Mw were detected by gel permeation chromatography (GPC). The thermal stability was evaluated by thermal gravimetric analysis (TGA), and their antioxidant activities were investigated including scavenging activity of superoxide and hydroxyl radical, reducing power and metal chelating ability. The results revealed that the introduction of quaternary ammonium groups and carboxymethyl groups decreased the crystallinity and the thermal stability of chitosan oligosaccharide (COS), and their antioxidant activities were closely related to the degree of substitution of quaternary ammonium groups and the carboxymethyl groups. This study provides important guidelines for developing new antioxidant agents.     

Sulfonated RAFT Copolymers as Heparin Mimetics: Synthesis,Reactivity Ratios,and Anticoagulant Activity

The glycosaminoglycan heparin is a clinically important anticoagulant drug, primarily used to reduce the risk of blood clots (thrombosis) during surgery. Despite its importance in medicine and its continuous use over many decades, heparin suffers from several limitations associated with its heterogeneity and its extraction from animal tissues. In order to address these limitations, reversible addition‐fragmentation chain transfer polymerization is utilized to prepare a library of heparin mimetic copolymers from the sulfonated monomers sodium 4‐styrene sulfonate, potassium‐3‐sulfopropyl acrylate, potassium‐3‐sulfopropyl methacrylate, and sodium‐2‐acrylamido‐2‐methyl‐1‐propane sulfonate. Copolymers are prepared using combinations of two different monomers in various ratios. Monomer reactivity ratios are also determined for some representative monomer combinations, and all polymers are characterized by ¹H NMR spectroscopy and gel permeation chromatography. The anticoagulant activities of the copolymers are determined by activated partial thromboplastin time and thrombin clotting time assays and structure–activity relationships are explored.     

Physicochemical Properties and Anticoagulant Activity of Purified Heteropolysaccharides from Laminaria japonica

Laminaria japonica is widely consumed as a key food and medicine. Polysaccharides are one of the most plentiful constituents of this marine plant. In this study, several polysaccharide fractions with different charge numbers were obtained. Their physicochemical properties and anticoagulant activities were determined by chemical and instrumental methods. The chemical analysis showed that Laminaria japonica polysaccharides (LJPs) and the purified fractions LJP0, LJP04, LJP06, and LJP08 mainly consisted of mannose, glucuronic acid, galactose, and fucose in different mole ratios. LJP04 and LJP06 also contained minor amounts of xylose. The polysaccharide fractions eluted by higher concentration of NaCl solutions showed higher contents of uronic acid and sulfate group. Biological activity assays showed that LJPs LJP06 and LJP08 could obviously prolong the activated partial thromboplastin time (APTT), indicating that they had strong anticoagulant activity. Furthermore, we found that LJP06 exerted this activity by inhibiting intrinsic factor Xase with higher selectivity than other fractions, which may have negligible bleeding risk. The sulfate group may play an important role in the anticoagulant activity. In addition, the carboxyl group and surface morphology of these fractions may affect their anticoagulant activities. The results provide information for applications of L. japonica polysaccharides, especially LJP06 as anticoagulants in functional foods and therapeutic agents.     

Structure and Anticoagulant Activity of a Galactofuranose-Containing Sulfated Polysaccharide from the Green Seaweed,Codium isthmocladum

A water-soluble sulfated polysaccharide, F2-1, was obtained from the marine green alga, Codium isthmocladum, using ion-exchange and size-exclusion chromatography. Structure analysis showed that the F2-1 was a sulfated arabinan comprising Ara, Rha, Man, Gal, and Xyl with an 18% sulfate content and a molecular weight of 100 kDa. Methylation analysis combined with desulfation, GC-MS, IR, and NMR spectroscopy showed that the backbone of F2-1 was →4)-β-L-Arap(1→ residue. Its 2-O and/or 3-O positions showed sulfate modification; additionally, the 2-O or 3-O position showed branch points. The side chains were composed of →5)-β-D-Galf, (1→2,6)-β-D-Galf(1→, (1→2)-β-L-Rhap4S, →4)-α-D-Glcp(1→, and terminal α-D-Galp(1→ and β-D-Xylp(1→. Polysaccharides containing β-D-galactofuranose are rarely found in seaweed. F2-1 exhibited significant anticoagulant activity in vitro. Our findings suggested that the green-tide alga, Codium isthmocladum, can be considered as a useful resource for bioactive polysaccharides.     

Antiplatelet and Anticoagulant Activity of Isorhamnetin and Its Derivatives Isolated from Sea Buckthorn Berries,Measured in Whole Blood

Blood platelets play a crucial role in hemostasis, the process responsible for keeping blood flowing in the circulatory system. However, unnecessary platelet activation can lead to aggregation at the site of atherosclerotic plaque rapture and the formation of a thrombus, which promotes atherothrombotic diseases. Various dietary components, such as phenolic compounds, are known to demonstrate antiplatelet and anticoagulant properties, and it is possible that these could form an important element in the prophylaxis and therapy of cardiovascular diseases. Our present study examined the biological activity of isorhamnetin (1) and two isorhamnetin derivatives, (2): 3-O-beta-glucoside-7-O-alpha-rhamnoside and (3): 3-O-beta-glucoside-7-O-alpha-(3″′-isovaleryl)-rhamnoside, isolated from the phenolic fraction of sea buckthorn fruit, against human washed blood platelets and human whole blood in vitro. The anti-platelet and anticoagulant potential was determined using (A) flow cytometry, (B) the thrombus-formation analysis system (T-TAS) and (C) colorimetry. The results of the T-TAS test indicate that the AUC₁₀ (Area Under the Curve) of the tested phenolic compounds (compounds 1, 2 and 3; 50 µg/mL) was markedly reduced compared to the control values. Moreover, flavonol demonstrated anti-platelet potential, including anti-adhesive activity, with these effects being more intense in compound 2 than isorhamnetin. Different actions of flavonol on platelet activation may depend on their binding ability to various receptors on blood platelets. However, the mechanism of their anti-platelet potential requires further additional studies, including in vitro and in vivo experiments.     

稀土三元配合物的低热固相合成、表征及抑菌活性研究

首次在室温条件下通过固相反应制得了镧稀土水杨酸8-羟基喹啉三元配合物La(Hsal)2(hq)(hq=C9H6NO^-;Hsal^-=C7H5COO^-)。采用元素分析、红外光谱、X射线粉末衍射、热重-差热分析等手段对配合物进行了表征。同时还初步分析了固相反应的机理。抑菌实验表明该配合物对大肠杆菌、金黄色葡萄球菌、白色念珠菌、芽孢杆菌都有很好的抑菌效果。     

肝素温度敏感色谱填料的制备与性能研究

利用自由基聚合法制备了含有聚(N-异丙基丙烯酰胺/N-丙烯酰氧琥珀酰亚胺)和肝素共聚物的温度敏感的亲和色谱填料; 通过热失重分析得到该填料表面聚合物的接枝率为9.45%; Elison-Morgan 法测定该填料表面的肝素含量为3.6mg/g. 将其用作HPLC 固定相, 分离苯和氢化可的松, 表明该色谱固定相具有温度敏感特性; 该色谱柱对凝血酶具有较强的亲和作用, 通过控制温度缩短了凝血酶的释放时间, 回收率为81.4%, 这为蛋白质的分离提供了一个快速有效的方法.