玉米芯木聚糖硫酸酯抗凝血及抗血小板聚集的活性研究

水不溶性木聚糖(wisX)经硫酸酯化获得玉米芯木聚糖硫酸酯wisX-S2、wisX-S4和wisX-S6,硫酸取代度(DS)分别为0.58、0.81和1.62,相对分子质量分别为1.1×105、1.3×105、1.0×105kDa。采用活化部分凝血活酶时间(APTT)、凝血酶时间(TT)、凝血酶原时间(PT)和血小板聚集实验检测了三者的抗凝血和抗血小板聚集活性。结果表明,wisX-S4和wisX-S6能明显延长APTT和TT,提示wisX-S是通过内源性和(或)共同途径发挥抗凝血作用的。同时wisX-S6体外能抑制血小板聚集。DS是影响wisX-S抗凝血活性的重要因素之一。     

褐藻多糖硫酸酯/甲基丙烯酸甲酯接枝聚合及抗凝血性能研究

研究了Cu(Ⅱ)离子存在时,褐藻多糖硫酸酯与甲基丙烯酸甲酯(MMA)的接枝聚合反应。主要探讨了V(MMA)/V(H2O)、Cu(Ⅱ)浓度、反应时间、温度对接枝率的影响。采用FT-IR对接枝共聚物进行了表征。接枝共聚物的体外抗凝血活性采用部分凝血活酶时间(APTT)、凝血活酶时间(PT)、凝血酶时间(TT)来评价。结果表明,褐藻多糖硫酸酯的水溶液在Cu(Ⅱ)离子存在时,可以引发MMA的自由基聚合反应。当褐藻多糖硫酸酯的质量浓度为5 6g/L,V(MMA)/V(H2O)=0 20,Cu(Ⅱ)浓度为3 26mmol/L,反应温度85℃,反应时间为3h时,接枝率为197.0%。APTT、TT、PT实验证明,接枝产物的APTT、TT比空白对照物分别延长20s和5s以上,具有抗凝血性能。     

丝素膜上接枝褐藻多糖硫酸酯及其体外抗凝血性能的研究

以戊二醛为交联剂,将褐藻多糖硫酸酯接枝在NH3等离子体处理的丝素膜的表面上。主要探讨了pH、反应温度、反应时间对丝素膜接枝效果的影响。用XPS分析了丝素膜表面化学元素。采用部分凝血活酶时间(APTT)、凝血活酶时间(PT)、凝血酶时间(TT)评价了接枝膜的体外抗凝血活性。得出最佳接枝工艺为:pH=2,反应温度40℃,反应时间6h。在该工艺条件下,接枝丝素膜APTT的最佳值为72s,APTT、TT凝血时间比空白对照分别延长37s和6s以上,具有抗凝血性能。经稳定性测试,固定的褐藻多糖硫酸酯基本上不脱落,从而获得了一种具有抗凝血性的新颖生物材料。     

淀粉硫酸酯复合工艺制备

以天然淀粉为原料，采用半干法酯化、交联复合工艺制备了具有抗凝血活性、低流度的硫酸酯淀粉钠，解决了硫酸化淀粉严重降解的难题，讨论了交联对产品流度及抗凝血活性的影响。     

抗凝血硫酸化微晶纤维素钠构效关系的初步研究

采用UV、IR、13CNMR和元素分析对3种由不同制备工艺参数所获得的硫酸化微晶纤维素钠(Na-MCS)进行结构分析,并研究其抗凝血活性。IR、UV揭示Na-MCS结构中含有硫酸酯基;13CNMR表明,3种Na-MCS的硫酸基取代模式相似,即C6取代占主导,C2位部分取代,C3位不发生取代;元素分析测得其硫酸取代度(DS)不同,即DSNa-MCS1=1 70、DSNa-MCS2=1 45、DSNa-MCS3=1 19;抗凝血研究表明,三者均可使血浆中活化部分凝血活酶时间(APTT)和凝血酶时间(TT)延长,但凝血酶原时间(PT)不延长,三者在抗凝血活性大小上存有差异,这与DS有关,在血浆中质量浓度为5μg/mL时,高DS的Na-MCS1使APTT达到155s、TT达34s,而低DS的Na-MCS3使APTT达到85s、TT达18s。构效关系分析表明,DS的差异不改变Na-MCS的抗凝血途径,但影响其抗凝血活性大小,DS越高,抗凝血活性越强。     

抗凝血硫酸化微晶纤维素的制备

以微晶纤维素(MC)为原料,采用氯磺酸-DMF硫酸化方法制备了具有抗凝血活性的硫酸微晶纤维素钠盐Na-MCS,并研究了以提高产物抗凝血活性为目标的工艺,即在常温条件下向原料MC中加入适量的浸润试剂DMF,并在真空下搅拌浸润;在盐水冰浴中制备氯磺酸-DMF酯化试剂;将适量酯化试剂加入已浸润的MC中搅拌反应;调节反应产物的pH值,在去离子水中透析,减压浓缩后真空干燥。     

纤维素硫酸酯的合成

以微晶纤维素为原料,以硫酸、正丙醇的混合物作为酯化剂,合成了纤维素硫酸酯,探讨了硫酸、正丙醇的摩尔比、液固比及反应时间影响因素,并以正交实验确定其最优工艺条件是：硫酸、正丙醇的摩尔比为1.95∶1,液固比是30∶1,反应时间为3 h,且最优工艺条件下可使酯化产物的收率稳定在97.31%左右。取代度能达到1以上。     

玉米芯木聚糖硫酸酯的合成、表征及其抗凝血活性

以碱法提取的玉米芯木聚糖为原料,LiCl为催化剂,在非质子溶剂中,采用三氧化硫-吡啶法合成了玉米芯木聚糖硫酸酯（XS）,通过正交试验对合成工艺进行了优化,并采用凝胶渗透色谱（GPC）、红外光谱（FT-IR）、元素分析和¹³C NMR对其结构与性能进行了表征,进一步考察了其体外抗凝血活性。结果表明：在DMF溶剂中,当三氧化硫-吡啶络合物与木糖羟基单元的物质的量之比为1.5：1时,于55℃反应3 h,木聚糖的酯化效果最好,所制备的XS的取代度（D_S）为1.53,得率为78.2%,重均相对分子质量为36 754,分散系数为1.191。结构表征发现,硫酸酯基已经成功引入木聚糖中。体外抗凝血活性结果表明：XS可以延长APTT和TT,具有一定的抗凝血活性;当取代度为1.53、质量浓度为20 mg/L时,XS的APTT、PT和TT分别为36.37、14.22和14.70 s,与阳性对照肝素钠基本相当。     

褐藻多糖硫酸酯

褐藻多糖硫酸酯是从海带中提取的纯天然药物,含有特效组分,具有抗凝血、降血脂、抗肿瘤和抗HIV的作用。通过对褐藻多糖硫酸酯药学、药理和毒理进行系统研究后,完成了Ⅱ期临床实验,发现它对治疗肾病综合症和早、中期慢性肾衰效果好,无毒副作用,特别对改善肾功能、降低血肌酐效果尤为明显。中国科学院海洋研究所在国内外首先将褐藻多糖硫酸酯用于临床治疗肾病综合症和早中期肾衰,将生物试剂制成为药物,形成了我国具有独立知识产权的海洋新药。     

褐藻多糖硫酸酯的生物活性及应用前景

褐藻多糖硫酸酯是褐藻的重要活性成分,可用于药品、保健品、食品和化妆品等领域。介绍了近年来褐藻多糖硫酸酯的生物活性研究进展,详细讨论了其抗凝血、抗肿瘤、免疫调节、抗炎等作用和工业应用前景。     

Distribution of cholesterol sulfate and its anabolic and catabolic enzymes in various rabbit tissues

Cholesterol sulfate (CS) recently has been shown to be involved in signal transduction pathway. To evaluate its functional significance, we determined the concentration of CS, and the specific activities of cholesterol sulfotransferase and CS sulfatase in various tissues of rabbit, and compared them with the concentration of sulfoglycolipids in rabbit tissues. CS was present in the epithelia and mucosa, but not in the tunica muscularis, of the digestive tract, trachea, uterine endometrium and uterine cervix. It was also present in lung, spleen, kidney, prostate, skin, hair, and nail at relatively high concentrations. Its concentration in the uterine endometrium was nine times higher in pseudopregnant rabbits than in nonpregnant rabbits because of activation of cholesterol sulfotransferase and inhibition of CS sulfatase in the pseudopregnant rabbits. Sulfoglycolipids were not detected in the uterine endometria of either nonpregnant-or pseudopregnant rabbits. However, sulfoglycolipids were detected at relatively high concentrations in the cerebrum, cerebellum, stomach, duodenum, jejunum, testis, and kidney of rabbits and thus the tissues in which both sulfolipids were detected were the gastrointestinal tract and kidney. In the digestive tract, the concentration of CS decreased in the order esophagus, stomach, duodenum, and jejunum, but that of sulfatide increased in the same order, indicating distribution of CS in the squamous epithelium. In addition, both CS and sulfatide were detected in the serum. On the other hand, CS sulfatase activity was detected in all tissues examined, even in hair, from which the enzyme was liberated by brief sonication, and its highest specific activity was deteted in the liver. The specific activity of cholesterol sulfotransferase varied among the tissues examined and was found to be significantly high in the esophageal epithelium and the uterine endometrium of pseudopregnant rabbit, indicating involvement of cholesterol sulfation in the formation of epithelium.     

Sulfation of silk fibroin by sulfuric acid and anticoagulant activity

Silk fibroin (Bombyx mori) was sulfated with an aqueous sulfuric acid solution. The sulfated fibroin was characterized by Fourier transform infrared spectroscopy (FTIR), amino acid analysis, and gel filtration chromatography (GFC). Maximum yield was obtained at around 2–4 h, and it decreased at 6 h and more. The molecular size decreased and dispersed with sulfation, and the molecular weight was estimated at around 10,000 by GFC using protein standards. The amino acid composition indicated that the crystal region of the fibroin molecule remained in sulfated fibroin until a sulfation reaction time of 4 h. The incorporation of sulfate groups was confirmed by FTIR and the amount of sulfate groups introduced for 4 h sulfation was estimated in 0.3 mmol/g by acidimetric titration. The efficiency of sulfation was calculated at 15.7%. Blood coagulation was prevented by 20 mg of sulfated fibroin in 1 mL of blood, while original fibroin did not show the effect. This result indicates that sulfate group introduction results in addition of anticoagulant function to silk fibroin. Although a variety of polymer backbones have been used for synthesis of sulfated polymers as anticoagulant materials, no reports are available concerning a sulfated polymer based on a protein backbone. Sulfated fibroin is a new type of anticoagulant material having a protein backbone. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 87: 2377–2382, 2003     

枸橼果实化学成分研究

研究枸橼Citrusmedica L.果实的化学成分,采用硅胶柱色谱、Sephadex LH-20、ODS等方法分离纯化化合物,应用波谱方法鉴定其结构。从枸橼果实中分离得到13个化合物,分别为:柠檬内酯(Ⅰ)、东莨菪亭(Ⅱ)、β-谷甾醇(Ⅲ)、奥巴叩酮(Ⅳ)、诺米林(Ⅴ)、柠檬苦素(Ⅵ)、异奥巴叩酸(Ⅶ)、橙皮素(Ⅷ)、胡萝卜苷(Ⅸ)、1-O-(β-D-葡萄糖基)-(2S,3S,4E,8E)-2-[(2′R)-2′-羟基十六酰氨基]-4(E),8(E)-十八二烯-1,3-二醇(Ⅹ)、柚皮素-7-O-β-D-葡萄糖苷(Ⅺ)、柚皮苷(Ⅻ)、橙皮苷(■)。     

Synthesis and spectroscopic analysis of cellulose sulfates with regulable total degrees of substitution and sulfation patterns via C NMR and FT Raman spectroscopy

Synthesis and spectroscopic characterisation of cellulose sulfate (CS) were reported. Various CS exhibiting diverse degrees of sulfation (DS_S) were prepared through acetosulfation or direct sulfation of cellulose. During the acetosulfation, intermediate product - cellulose acetate sulfate (CAS) - was formed after the comparative esterification and subsequent deacetylation of CAS led to CS. The direct sulfation proceeded quasi-homogeneously and heterogeneously in N,N-dimethylformamide (DMF) or homogeneously in N,N-dimethylacetamide (DMAc)/LiCl mixture. The total DS_S between 0.21-2.59 and partial DS_S6 as well as DS_S2 of up to 1 were determined via elemental analysis and ¹³C NMR spectroscopy. Besides, solid-state CP/MAS ¹³C NMR could characterise CS regarding the sulfation. Subsequently, FT Raman investigation of obtained CS was conducted with the aim to establish analysis methods quantifying the total DS_S. The intensities of Raman bands ascribed to the vibrations of OSO and C-O-S groups were used as analysis parameters, yielding calibration curves with high correlation coefficients of more than 0.96.     

去冷沉淀血浆的质量分析

目的比较去冷沉淀血浆(Cryoprecipitate-reduced plasma,CRP)中系列凝血因子、纤维蛋白原(Fibrinogen,Fib)和血管性血友病因子裂解蛋白酶(A disintegrin-like and metalloprotease with thrombospondin-1 repeats 13,ADAMTS-13)水平的变化。方法用140人份新鲜冰冻血浆(Fresh frozen plasma,FFP)制备CRP,采用Biggers一期法测定FFP和CRP中FⅡ、FⅤ、FⅦ、FⅧ、FⅨ、FⅩ、FⅪ、FⅫ的促凝活性;免疫比浊法测定Fib和血管性血友病因子抗原因子活动度(vWF∶Ag)水平;荧光共振能量转移法(Fluorescence resonance energy transfer,FRET)测定70人份FFP和CRP中的ADAMTS13活性及抗原含量。结果与FFP比较,CRP中FⅧ∶C、FⅤ∶C、FⅦ∶C、FⅨ∶C、FⅩ∶C、FⅪ∶C、Fib、vWF∶Ag水平均明显下降,且差异均有统计学意义(P<0.05或<0.001);FⅡ∶C、FⅫ∶C、ADAMTS13抗原含量和活性差异均无统计学意义(P>0.05)。结论 CRP可代替FFP用于血栓性血小板减少性紫癜(Thrombotic throm-bocytopenic purpura,TTP)的治疗,而不适用于FⅧ、Fib及vWF缺乏患者的补充治疗。     

Investigations on synthesis and characterization of carboxy-groups containing cellulose sulfates

Summary Carboxymethyl cellulose and C-6-carboxy cellulose were treated with SO₃ and HSO₃CL in DMF yielding the corresponding watersoluble sulfate half-esters. A preferred sulfation of primary hydroxyl groups was found by 13C-n. m.r. spectroscopy. The potentiometric titration of the polyelectrolytes showed significant differences in the titration behaviour which can be explained by the different acidity of the carboxy groups in the two cellulose derivatives.     

Keratanase II-catalyzed synthesis of keratan sulfate oligomers by using sugar oxazolines as transition-state analogue substrate monomers: a novel insight into the enzymatic catalysis mechanism

Keratan sulfate (KS) oligomers with well-defined structures were synthesized by keratanase II (KSase II)-catalyzed transglycosylation. N-Acetyllactosamine [Galbeta(1-->4)GlcNAc; LacNAc] oxazoline derivatives with sulfate groups at the C-6 (1 a) and both the C-6 and the C-6' (1 b) were prepared as transition-state analogue substrate monomers for KSase II. Monomer 1 a was effectively oligomerized by the enzyme under weak alkaline conditions, to give alternating 6-sulfated KS oligomers (2 a) in good yields, and with total control of regioselectivity and stereochemistry. KSase II also recognized 1 b, which provided fully 6-sulfated KS oligomers (2 b) in good yields under similar conditions. Nonsulfated LacNAc oxazoline was difficult to oligomerize enzymatically. These results imply that the catalysis mechanism of KSase II involves a sugar oxazolinium ion that requires the 6-sulfate group in the GlcNAc residue not only in hydrolysis of KS chains, but also in oligomerization of oxazoline monomers. This is the first report of KSase II-catalyzed transglycosylation to form beta(1-->3)-glycosidic bond through a substrate-assisted mechanism.     

NMR and FT Raman characterisation of regioselectively sulfated chitosan regarding the distribution of sulfate groups and the degree of substitution

Chitosan sulfates (CHS) were prepared with chlorosulfonic acid homogeneously and non-homogeneously. The total degrees of substitution (DS) ascribed to sulfate groups (DS_S) were determined with elemental analysis and the partial DS at 6-O-position was estimated via ¹³C NMR. CHS with diverse total DS_S and sulfation patterns were obtained according to the analysis. The effects of selected reaction parameters that can influence the distribution of sulfate groups were examined. The structure of CHS was then characterized with various NMR techniques, i.e. one- (1D-) and two-dimensional (2D-) NMR, and FT Raman spectroscopy. It was found that the primary hydroxyl groups were always predominantly sulfated for CHS prepared under homogeneous or non-homogeneous conditions and no sulfate groups at 2-N-position could be detected. Finally, the feasibility of using FT Raman spectroscopy as another alternative for determining the total DS_S of CHS was presented.     

硼酸酯催化合成2-羟基-4-甲氧基二苯甲酮

以2,4 二羟基二苯甲酮及硫酸二甲酯为原料,用自制的N 正辛基硼酸二乙醇胺酯为催化剂合成了2 羟基 4 甲氧基二苯甲酮,研究了原料摩尔比、催化剂用量、反应时间、反应温度等因素对产品的影响。实验表明,在n(2,4 二羟基二苯甲酮)∶n(硫酸二甲酯)=1∶0 55,m(N 正辛基硼酸二乙醇胺酯)/m(2,4 二羟基二苯甲酮)=0 02,反应温度70℃,反应时间4h的条件下,w(2 羟基 4 甲氧基二苯甲酮)=98 8%,w(2,4 二甲氧基二苯甲酮)<0 8%,收率达94 6%。