昆布多糖硫酸酯化修饰及抗肿瘤活性的研究

对昆布多糖进行硫酸酯化修饰,考察修饰前后多糖结构及抗肿瘤活性的变化。采用氯磺酸-吡啶法进行多糖硫酸酯化修饰,考察了昆布多糖及其硫酸酯的红外光谱、核磁光谱特征,扫描电镜观察了表面形态,采用MTT比色法进行抗肿瘤活性评价。结果表明,昆布多糖及其硫酸酯都具有典型的多糖红外吸收,昆布多糖硫酸酯具有硫酸基的特征吸收峰;昆布多糖及其硫酸酯均是以β-(1→3)糖苷键为主链的多糖,昆布多糖硫酸酯的硫酸基取代位置在C2-OH与C6-OH。昆布多糖及其硫酸酯表面立体形态差异显著,昆布多糖表面呈云雾状或海绵状,昆布多糖硫酸酯表面呈片状或块状。昆布多糖及其硫酸酯对人肠癌细胞LOVO生长都具有明显的抑制作用,并且昆布多糖硫酸酯的抗肿瘤作用强于昆布多糖。     

薤白多糖的硫酸化修饰及体外抗氧化活性

为探讨薤白多糖硫酸化修饰的最佳条件,以及硫酸化修饰提高薤白多糖活性的可能性,采用氯磺酸-吡啶法对醇沉法得到的薤白多糖和柱层析纯化的3种分级薤白多糖进行硫酸化修饰,以氯磺酸-吡啶配比、反应温度和反应时间为自变量,修饰产物的硫酸基取代度(DS)为响应值,应用响应面设计法确定硫酸化修饰的最佳条件,用H2O2/Fe2+体系法和邻苯三酚自氧化法测定修饰产物的抗氧化活性。结果表明:薤白多糖氯磺酸-吡啶法修饰的最佳条件为氯磺酸∶吡啶=1∶3,反应温度65℃,反应时间2 h,此条件下硫酸根取代度为0.470,硫酸化修饰能提高薤白多糖的体外抗氧化活性。     

硫酸酯化银耳多糖的制备及抗氧化性活性研究

采用氯磺酸-吡啶法化学合成硫酸酯化银耳多糖,对硫酸酯化反应的吡啶与氯磺酸的体积比进行优化,氯化钡-明胶比浊法测定硫酸酯化银耳多糖的取代度,并对不同取代度的硫酸酯化银耳多糖进行体外抗氧化实验。结果表明:硫酸酯化银耳多糖取代度随吡啶与氯磺酸体积比的提高而增加;当吡啶与氯磺酸体积比为4∶1,反应温度60℃,反应时间3 h时,产物的取代度为1.32,在0.8～2.0 mg/mL浓度范围内对Fenton反应产生的羟基自由基体外清除率相对于银耳多糖具有明显优势。     

香菇多糖的化学修饰及结构表征

为明确改性效果,本文选择适宜的方法对香菇多糖分子结构进行了化学修饰和结构表征.选用氯磺酸-吡啶法和浓硫酸法对香菇多糖硫酸酯化,并进行了比较,利用一氯乙酸法对其进行羧甲基化,借助红外光谱和核磁共振碳谱对样品分别进行了结构表征.结果表明,氯磺酸-吡啶法硫酸化取代度达1.38,产率达79.23%,硫含量达14.59%,效果优于浓硫酸法;1247和807 cm~(-1)的特征吸收峰证明了硫酸基的引入,碳谱δ 79.06附近的峰证明了C2和C4被部分硫酸基取代;羧甲基化碳谱中C2和C4位附近出峰说明其位上羟基被取代,且异头碳为单一β-D-吡喃环糖苷键构型.通过红外光谱和核磁共振碳谱对香菇多糖结构表征证实,硫酸酯化和羧甲基化二种化学修饰方法对香菇多糖分子结构改性是可行的,且氯磺酸-吡啶法更适合香菇多糖硫酸化.     

硫酸酯化川芎多糖的制备及其抗氧化活性研究

以三种川芎多糖组分和淀粉为原料采用氯磺酸-吡啶法制备硫酸酯化产物,紫外和红外光谱对其进行结构表征,氯化钡-明胶比浊法测定硫酸酯化川芎多糖的取代度,分别考察试样在硫酸酯化前后对邻苯三酚自氧化反应产生的超氧阴离子和1,1-二苯基-2-苦基肼(DPPH)自由基的体外清除率。实验结果表明:川芎粗多糖和硫酸酯化川芎多糖对两种自由基的清除作用远低于阳性对照维生素C,但硫酸酯化修饰有助于提升川芎多糖对DPPH自由基的清除作用,对超氧阴离子自由基无明显影响;硫酸基的引入能够提高淀粉对O2-.的清除作用,但清除作用很弱。     

几种硫酸化试剂和溶剂对菊糖硫酸化改性的影响

为制取硫酸化菊糖,以硫酸钡比浊法测定硫酸基取代度(DS)、红外光谱测定含硫基团的特征吸收峰、核磁共振碳谱(13C NMR)判断硫酸根取代位置等方法,比较了以N,N-二甲基甲酰胺(DMF)、二甲基亚砜(DMSO)和吡啶(Py)三种溶剂,氯磺酸(CA)和三氧化硫(SO3)两种硫酸化试剂对菊糖硫酸酯化的影响.结果表明:以吡啶为溶剂、氯磺酸为硫酸化试剂的方法(CA-Py)与SO3-Py、CA-DMF三种硫酸化方法均获得了硫酸化菊糖,产品均显示不对称S=O键伸缩振动(约1255 cm-1)和对称的C-O-S键伸缩振动(约810 cm-1)特征吸收峰;三种方法的DS分别为:1.24,0.89,1.83;三种产品的13C NMR基本相同,均表明硫酸根连接在C3、C5、C6上.DMSO不适宜用作硫酸化溶剂.三种硫酸化方法是成功的,但以SO3-Py法操作简便,最适于菊糖硫酸化.     

响应面法优化猴头菌硫酸化多糖

[目的]为了获得猴头菌多糖的最佳硫酸化修饰条件,提高猴头菌硫酸化多糖硫酸基团的取代度。[方法]采用单因素比较氯磺酸-吡啶摩尔比、反应温度和反应时间对猴头菌硫酸化多糖取代度的影响,利用响应面法对各因素的最佳水平以及各因素之间的交互作用进一步研究。[结果]猴头菌多糖的最佳硫酸化修饰条件为:氯磺酸-吡啶摩尔比为1:4、反应温度为59℃、反应时间为2. 6 h,取代度为0. 457,与模型的预测值基本相符。[结论]响应面法优化得到猴头菌多糖的硫酸化修饰条件参数准确,该模型可以用于猴头菌多糖硫酸化修饰条件的优化。     

响应面法探讨氯磺酸-吡啶法修饰条件对硫酸化当归多糖取代度的影响

用氯磺酸-吡啶法对当归多糖进行硫酸化修饰。用响应面法研究了修饰条件中反应温度(A)、反应时间(B)和氯磺酸-吡啶比例(C)三个因素对产物的硫酸基取代度的影响,建立回归模型,验证了其有效性,并分析了主效应和因素交互作用。结果表明,在A为65~95℃、B为60~180 m in、C为1:3~14.3范围内,三个因素与产物平均取代度(Y)的回归模型为Y=2.74+0.66×A+0.66×B+0.80×C+0.51×A×B-0.31×A×C-0.16×B×C-0.34×A2-0.23×B2-0.50×C2;F检验证明模型拟合较好,可以用于量化控制反应条件;三个因素对取代度的影响程度为CA=B,A和B之间存在极显著的交互作用。     

硫酸酯化剂和溶剂对海参岩藻聚糖硫酸酯化修饰的影响

比较了以N,N-二甲基甲酰胺(DMF)和吡啶(Py)两种溶剂,氯磺酸-吡啶(CA-Py)和三氧化硫-吡啶(SO3-Py)两种硫酸酯化剂对海参岩藻聚糖硫酸酯化的影响。结果表明:以CA-Py为酯化剂可使硫酸基的含量高达50%以上,以SO3-Py为酯化剂,硫酸基的含量仅达到20%左右;DMF为溶剂时可低温反应,Py为溶剂时反应温度必须90℃以上,但产品得率较高。红外光谱分析显示:硫酸酯化后,四种产品硫酸酯的特征吸收峰均显著增强。抗氧化活性实验结果表明:四种硫酸酯化产品对1,1-二苯基-苦肼基自由基的清除效果最好,对羟基自由基的清除效果次之,对超氧阴离子自由基的清除效果最小;以SO3-Py为酯化剂所得产品的抗氧化活性优于CA-Py为酯化剂的产品,DMF为溶剂的产品优于Py为溶剂的产品。因此,操作简便的SO3-Py法更适于海参岩藻聚糖的硫酸酯化。     

魔芋低聚糖硫酸酯的制备及抗病毒活性研究

从酶解魔芋干粉中提取分子量在1500左右的葡甘露低聚糖Apkos,采用吡啶-氯磺酸法制备了它的硫酸酯衍生物S-Apkos,其硫含量为8.28%,取代度(Ds)为0.57.用Vero细胞和单纯疱疹病毒2型(HSV-2)考察硫酸酯化前后低聚糖的抗病毒活性.结果表明,本身无抗HSV-2活性的Apkos硫酸酯化后产生了抗病毒活性,500～4000μg/ml的SApkos能很好地抑制HSV-2引起的Vero细胞病变,而在500μg/ml及以上浓度条件下能完全抑制HSV-2在Vero细胞单层中形成病毒空斑.     

Barley leaf peroxidase: purification and characterization

Peroxidase was prepared from extracts of barley leaves and separated into seven components, different in pI. The purification procedure comprised two parts. The first part was based on the fact that all the components had practically the same molecular weights. It consisted of fractionations with acetone and ammonium sulfate, ion-exchange chromatographies on CM-cellulose and DEAE-Sepharose CL-6B, and molecular-sieve chromatography on Ultrogel AcA44; the components were all purified together to near homogeneity on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and the procedure resulted in 1,200-fold purification with a yield of 39%. The ion-exchange chromatographies were carried out under conditions such that the components would not be adsorbed. In the second part, the enzyme preparation was separated into the seven components by repeating isoelectric electrophoresis. Their isoelectric points (pI) were 6.3, 6.8, 7.4, 8.3, 8.5, 8.7, and 9.3. The components other than the pI 6.3 and 6.8 components were each purified to homogeneity in the electrophoresis. The seven components thus prepared were the same in molecular weight on SDS-gel electrophoresis (44,000) and showed absorption maxima at the same wave-lengths (403, 496, and 534 nm), RZ (A403/A275) ranging from 2.09 to 2.81. Their protoheme IX contents were 0.81-1.07 mol/mol, and their true sugar contents 15-26% (g/g). The amino acid compositions suggest that the five components described above are not real isoenzymes, but exhibit different pI values due to differences in glycosyl residue. The pI 9.3 component was crystallized in spite of its high sugar content.     

Preparation of Acetylated Grapefruit Peel Polysaccharide

Grapefruit peel polysaccharide has antioxidant, antitumor, hypoglycemic and other biological activities, and chemical modification can further improve the properties of the polysaccharide. Acetylation modification of polysaccharides has the advantages of simple operation, low cost and little pollution, and is widely used at present. Different degrees of acetylation modification have different effects on the properties of polysaccharides, so it is necessary to optimize the preparation technology of acetylated grapefruit peel polysaccharides. In this article, acetylated grapefruit peel polysaccharide was prepared by acetic anhydride method. With the degree of acetyl substitution as the evaluation index, combined with the analysis of sugar content and protein content in the polysaccharide before and after modification, the effects of three feeding ratios of 1:0.6, 1 : 1.2 and 1 : 1.8 (polysaccharide: acetic anhydride, mass/volume) on acetylation modification were explored through single factor experiments. The results showed that the optimum ratio of material to liquid for acetylation modification of grapefruit peel polysaccharide was 1:0.6. Under these conditions, the degree of substitution of acetylated grapefruit peel polysaccharide was 0.323, the sugar content was 59.50 % and the protein content was 1.038 %. The results provide some reference for the study of acetylated grapefruit peel polysaccharide.     

Anticoagulant and antithrombin activities of oversulfated fucans.

Three species of oversulfated fucans having different sulfate contents (the ratio of sulfate/total sugar residues, 1.38-1.98) were prepared by chemical sulfation of a fucan sulfate (sulfate/sugar ratio, 1.28) isolated from the brown seaweed Ecklonia kurome. The anticoagulant activities of the oversulfated fucans were compared with that of a parent fucan with respect to activated partial thromboplastin time (APTT) and thrombin time (TT) in plasma. The respective activities (for APTT and TT) of the oversulfated fucans increased to 110-119% and 108-140% of the original values with increase in their sulfate content. The anticoagulant activity with respect to APTT (173 units/mg) of an oversulfated fucan (sulfate/sugar ratio, 1.98) was higher than that (167 units/mg) of heparin used as a standard. The heparin cofactor II-mediated antithrombin activity of the oversulfated fucans also increased significantly with increase in sulfate content. The maximum activity was higher than those of the parent fucan and heparin. However, the increment of the anticoagulant and the antithrombin effects gradually decreased with increase in the sulfate content of the fucans. These results indicate that the effects of the fucan sulfate are dependent on its sulfate content until a plateau is reached.     

Sulfated modification of the water-soluble polysaccharides from Polyporus albicans mycelia and its potential biological activities

In this study, three chemically sulfated polysaccharides (SPAPs) were derived from one water-soluble polysaccharide (PAP) of Polyporus albicans mycelia by chlorosulfonic acid-pyridine method. The effects of polysaccharides on the immune function were examined after the mice were intragastrical administrated with polysaccharides at three doses of 100, 200, and 300 mg/kg body weight for 7 days. The results showed that both the lymphocytes proliferation and macrophage function were significantly enhanced by SPAP in all groups along with the increase of the substitution degree and dose (P<0.01). It indicated that SPAP could be a potential immunostimulants used in the food and pharmaceutical industry.     

Purification and properties of beta-D-glucosidase (linamarase) from the butter bean, Phaseolus lunatus

A beta-D-glucosidase (linamarase) was purified 11,700-fold from the butter bean, Phaseolus lunatus L., by means of successive procedures including extraction, ammonium sulfate fractionation, acetone treatment, and chromatographies on CM-Sephadex, DEAE-Sephadex, and Sephadex G-200. The final preparation gave a single protein band on both disc polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. In spite of its electrophoretic purity, the final enzyme preparation showed four glycosidase activities; beta-D-glucosidase, beta-D-galactosidase, beta-D-fucosidase, and beta-D-xylosidase. The molecular weight of the enzyme was determined to be 124,000 +/- 9,000 by Sephadex G-200 gel filtration, and 59,000 +/- 2,400 by SDS-disc gel electrophoresis. The enzyme showed a pH optimum in the range of 5.1 to 6.0 with p-nitrophenyl beta-D-glucoside, 4-methylumbelliferyl beta-D-glucoside, and linamarin. Among natural substrates containing a beta-glucosyl terminal, linamarin, prunasin, and salicin were hydrolyzed by the enzyme from butter beans, but amygdalin, cellobiose, gentiobiose, and laminarin were hardly hydrolyzed.     

Purification and properties of glycine N-methyltransferase from rat liver

Glycine N-methyltransferase (EC 2.1.1.20) has been purified to homogeneity from rat liver. The enzyme has a molecular weight of 132,000 by sedimentation equilibrium method. This value is in good agreement with a value of 130,000 obtained by Sephadex G-150 chromatography. The molecular weight of the denatured enzyme as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate is 31,500. The numbers of peptides obtained by tryptic digestion and by cyanogen bromide cleavage are one-fourth of those expected from the contents of lysine plus arginine residues and methionine residues, respectively. By Edman degradation, phenylthiohydantoin-leucine is the only amino acid derivative released from the enzyme. Neither sugar nor phospholipid is detected in the purified preparation. These data indicate that the rat liver glycine N-methyltransferase is a simple protein consisting of 4 identical subunits. The enzyme has an isoelectric pH of 6.4, and is most active at pH 9.0. From the circular dichroism spectrum, an alpha helix content of about 11% is calculated. Whereas the initial velocity as a function of glycine concentration gives a Michaelis-Menten kinetics, the enzyme shows a positive cooperativity with respect to S-adenosylmethionine. The concentrations of glycine and S-adenosylmethionine which give a half-maximum velocity are 0.13 mM and 30 microM, respectively, at pH 7.4 and 25 degrees C.     

Purification and characterization of poly(ADP-ribose) synthetase from calf thymus.

Poly(ADP-ribose) synthetase from calf thymus has been purified to apparent homogeneity by a simple and rapid method with a recovery of 10 to 20%. The enzyme activity absolutely requires the presence of DNA. Histone further stimulates the reaction. The Km for NAD and the maximal velocity at 25 degrees C and pH 8.0 in the presence of both compounds are 55 micron and 1,400 nmol/min/mg, respectively. The sedimentation coefficient (s020,w) of the enzyme is 5.80 S. The molecular weight is calculated to be 108,000 by sedimentation equilibrium method using a partial specific volume of 0.736 ml/g. This value is in good agreement with the molecular weight values of 115,000 and 120,000 determined by gel filtration on Sephadex G-200 and gel electrophoresis in the presence of sodium dodecyl sulfate, respectively. The enzyme is colorless and its absorption spectrum shows a maximum at 280 nm. From a CD spectrum, alpha helical content is estimated to be approximately 30%. The enzyme is a basic protein having a pI value of 9.8 and is rich in lysine rather than arginine. Neutral sugar, phospholipid, and DNA are not detected in the final preparation. These data indicate that the purified enzyme is a simple globular protein composed of a single polypeptide having an approximate molecular weight of 110,000.     

Bulk Preparation of CNS Cytoskeleton and the Separation of Individual Neurofilament Proteins by Gel Filtration: Dye-Binding Characteristics and Amino Acid Compositions

Abstract: The three major proteins of mammalian neurofilaments, of molecular weight 70,000, 160,000, and 210,000, have been resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel eJectrophoresis, and more recently, by ion-exchange chromatography in urea solution. We describe here a method to separate the neurofilament proteins by gel filtration without the use of SDS. A bulk preparation of cytoskeleton from rat spinal cord was first characterized. This preparation was then solubilized in a buffer containing 8 M urea and subjected to gel filtration. Individual neurofilament proteins, in milligram quantities, were harvested following the pooling of appropriate fractions. Gel electrophoresis showed a high degree of homogeneity in each of the three pooled fractions. Dye binding studies demonstrated that the protein of molecular weight 210,000 was relatively underrepresented when stained with Coomassie Blue, while all three neurofilament proteins showed similar dye binding properties with Fast Green. Amino acid analysis indicated that (1) all three neurofilament proteins contained a high content of acidic residues; (2) the molecular weight 210.000 protein contained >8 mol% proline; and (3) no simple oligomeric relationship existed among the neurofilament triplets.     

Purification,properties and comparison of invertase,exoinulinases and endoinulinases of Aspergillus ficuum

Summary One invertase (Inv), five exoinulinases (Exo I; II; III; IV; V) and three endoinulinases (Endo I; II; III) were isolated from a commercial inulinase preparation derived from Aspergillus ficuum using ammonium sulfate precipitation, ion exchange chromatography on DEAE-Sephacel and DEAE-Trisacryl, gel filtration on Ultrogel and Fast Protein Liquid Chromatography on a Mono Q column. The invertase (Inv) had a molecular weight of 84000 and was much more active on sucrose than on inulin: the ratio of activity on inulin and sucrose (I/S ratio) was 0.01. The five exoinulinases show the same molecular weight of 74000 and I/S ratios in the range 0.16–0.36. The three endoinulinases had molecular weight of 64000 and I/S ratios in the range 0.86–2.92. All the -fructofuranosidases were glycoproteins with a high sugar content (from 22 to 41% w/w). A. ficuum is the first described organism containing the three activities: invertase, exo and endoinulinase.