白薇化学成分及其抗烟草花叶病毒活性研究

前期研究发现seco-pregnane类甾体苷具有较强的抗烟草花叶病毒(TMV)活性,为进一步寻找活性化学成分,开展白薇化学成分研究。从白薇乙醇提取物的氯仿部位中分离得到10个单体化合物,根据其理化性质以及波谱数据鉴定为:glaucogenin-C 3-O-α-L-diginopyranosyl-(1→4)-β-D-thevetopyranoside(1)、glaucogenin-C 3-O-β-D-oleandropyranosyl-(1→4)-β-D-digitoxopyranosyl-(1→4)-α-L-cymaropyranoside(2)、glaucogenin-C 3-O-β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-β-D-oleandropyranoside(3)、glaucogenin-A 3-O-α-L-cymaropyranosyl-(1→4)-β-D-digitoxopyranosyl-(1→4)-β-D-oleandropyranoside(4)、glaucogenin-A 3-O-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-D-oleandropyranoside(5)、glaucogenin-A 3-O-α-L-cymaropyranosyl-(1→4)-α-L-cymaropyranosyl-(1→4)-β-D-oleandropyranoside(6)、glaucogenin-A 3-O-α-L-cymaropyranosyl-(1→4)-β-L-cymaropyranosyl-(1→4)-β-L-cymaropyranoside(7)、glaucogenin-A 3-O-α-L-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-β-L-cymaropyranoside(8)、antofine(9)、2-O-β-D-fructofuranosyl-β-D-glucopyranoside(10)。化合物1～8,10均为首次从该植物中分离得到。采用半叶枯斑法,从钝化活性、保护活性、治疗活性三方面评估化合物1～9的生物活性,结果表明,化合物1和9具有显著的抗TMV活性。     

黑刺菝葜中的甾体皂苷

从黑刺菝葜(Smitax scobinicaulis C.H.Wringh)根茎中再次分离得到2个新的甾体皂苷化合物,经理化、光谱分析及与标准样品对照,鉴定化合物Ⅲ为(25D)螺甾-5-烯-3β,17α,27-三羟基-3-O-β-D-吡喃葡萄糖-(1→4)-O-[α-L-吡喃阿拉伯糖(1→6)]-β-D-吡喃葡萄糖甙。化合物Ⅳ为(25D)螺甾-3β,17α,27-三羟基-3-O-β-D-吡喃葡萄糖-(1→4)-O-[α-L-吡喃阿拉伯糖(1→6)]-β-D-吡喃葡萄糖甙。     

排风藤中皂苷类化学成分研究

从茄属植物排风藤的全草中分离得到了4个皂苷类化合物,经鉴定分别为:25R-螺甾-3-O-[β-D-吡喃木糖基-(1→3)]-O－β-D-吡喃葡萄糖基-(1→2)-O-β-D-吡喃葡萄糖基-(1→4)-O-β-D-吡喃半乳糖苷(1),5α,25R-螺甾-3-O-[β-D-吡喃木糖基-(1→3)]-O-β-D-吡喃葡萄糖基-(1→2)-O－β-D-吡喃葡萄糖基-(1→4)-O-β-D-吡喃半乳糖苷(2),22α,25R-26-O-β-D-吡喃葡萄糖基-22-羟基-呋甾-△5-3β,26-二醇-3-O-β-D-吡喃葡萄糖基-(1→2)-O-[β-D-吡喃木糖基-(1→3)]-O-β-D-吡喃葡萄糖基-(1→4)-O-β-D-吡喃半乳糖苷(3),22α,25R-26-O-β-D-吡喃葡萄糖基-22-羟基-呋甾-△5-3β,26-二醇-3-O－β-D-吡喃葡萄糖基-(1→2)-O-β-D-吡喃葡萄糖基-(1→4)-O-β-D-吡喃葡萄糖苷(4).化合物1-4均为首次从排风藤中分离得到.     

褐藻胶寡糖生物活性研究进展

褐藻寡糖AOS由β-D-甘露糖醛酸(ManA)和α-L-古洛糖醛酸(GulA)通过1-4糖苷键连接而成,具有广泛的生物活性,如促进植物生长、缓解植物非生物胁迫、抗肿瘤、抑菌等作用,在绿色农业、医药保健等领域具有广阔的前景。本文综述了近几年来褐藻胶寡糖在生物活性方面的研究进展,并对其应用前景进行了展望。     

羊栖菜褐藻糖胶部分酸水解产物中寡糖的结构分析

研究了羊栖菜褐藻糖胶DSF32部分酸水解小分子产物中寡糖的结构。DSF32经0.5 mol/L三氟乙酸(TFA)部分酸水解,水解液用截留分子量为3500 Da的透析袋进行透析,然后用D201柱将透过液分为中性糖部分(05N)和酸性糖部分(05A)。采用液质联用和甲基化分析研究了它们的结构,结果表明,05A可能存在以下寡糖:→4)G lcA(1→2)Hex(1→,→2)Hex(1→4)G lcA(1→2)Hex(1→,→4)G lcA(1→2)Hex(1→3)Fuc(1→。05N的一个组分05N-3的非还原末端是Hex(1→,还原末端是2→)Hex,寡糖中间的糖苷键类型主要是1,6,另外还有少量的1,4和1,2,6连接方式。     

西南远志中一个新的三萜皂苷北大核心CSCD

从西南远志根中分离得到3个齐墩果酸型皂苷类化合物,根据理化性质和波谱数据鉴定其结构分别为3-O-β-D-葡萄糖基presenegenin 28-O-α-L-阿拉伯糖基-(1→3)-β-D-木糖基-(1→4)-[β-D-芹糖基-(1→3)]-α-L-鼠李糖基-(1→2)-[β-D-葡萄糖基-(1→3)]-[4-O-(E/Z)-3″,4″,5″-三甲氧基肉桂酰基]-β-D-岩藻糖基酯(1)、3-O-β-D-葡萄糖基presenegenin 28-O-β-D-木糖基-(1→4)-α-L-鼠李糖基-(1→2)-[α-L-鼠李糖基-(1→3)]-[4-O-(E/Z)-对甲氧基肉桂酰基]-β-D-岩藻糖基酯(2)和3-O-β-D-葡萄糖基presenegenin 28-O-α-L-阿拉伯糖基-(1→4)-β-D-木糖基-(1→4)-[β-D-芹糖基-(1→3)]-α-L-鼠李糖基-(1→2)-[4-O-(E/Z)-对甲氧基肉桂酰基]-[α-L-鼠李糖基-(1→3)]-β-D-岩藻糖基酯(3),其中化合物1为新化合物,化合物2和3首次从该植物中分离得到。     

仙茅中两个新的环阿尔廷醇型三萜皂苷

从仙茅(Curculigo orchioides）中分离得到2个新的环阿尔廷醉型三萜皂苷,通过波诺分析鉴定了它们的化学结构,即3β,11α,16β—三羟基环阿尔廷烷-24-酮-3-O-[β-D-吡喃葡糖(1→3)-β-D-吡喃葡糖(1→2)-β-吡喃葡糖]-16-O-α-L-阿拉伯糖苷(1)和(24S)-3β,11α,16β,24-四经基环阿尔廷烷-3-O-[β-D-吡喃葡糖(1→3)-β-D-吡喃葡糖(1→2)-β-D-吡喃葡糖]-24-O-β-D-吡喃葡糖苷(2)。     

白首乌甙A,B和C的结构

从著名中药白首乌(Cynanchum auricutatum Royle ex Wight)根中分离得到7个C_2(?)体甙。其小4个已知物——wilfosidc C3N (Ⅰ), C1N (Ⅱ), C1G (Ⅲ), K1N (Ⅴ),和另外3个新C_(21)甾体甙,命名为白首乌甙A (Ⅵ),B (Ⅶ),C(Ⅳ) (cynauricusidc A, B,C)。经光谱数据分析和化学反应,证明其结构分别为:开德甙元 3-氧-β D-葡萄糖吡喃基-(1→4)-α-L-加拿大麻糖吡喃基-(1→4)-β-D-加拿大麻糖吡喃基-(1→4)-α-L-迪吉糖吡喃基-(1→4)-β-D-加拿大麻糖吡喃甙(kidjoranin 3-O-β-D-glucopyranosyl-(1→4)-α-L-cymaropyranosy-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-D-cymaropyranoside, Ⅵ);萝藦甙元 3-氧-α-L-加拿大麻糖吡喃基-(1→4)-β-D-加拿大麻糖吡喃基-α-L-迪吉糖吡喃基-(1→4)-β-D-加拿大麻糖吡喃甙(metaplexigenin -O-α-L-cymaropyranosyl-(1→4)-β-D-cymaropyranosyl-(1→4)-α-L-diginopyranosyl-(1→4)-β-cymaropyranoside,Ⅶ);告达庭 3-氧-β-D-葡萄糖吡喃基-(1→4)-β-L-葡萄糖吡喃基-(1→4)-α-L-加拿大麻糖吡喃基-(1→4)-β-D-加拿大麻糖吡喃基-(1→4-α-L-迪吉糖吡喃基-(1→4)-β-D-加拿大麻糖吡喃基(caudatin 3-O-β-D-glucopyranosy-(1→4)-β-D-glucopyr     

滇重楼地上部分的甾体皂甙

从滇重楼Paris polyphylla Sm. var. yunnanensis (Fr.) H-M.地上部分分离得到3个甾体皂甙,经光谱测定和化学降解证明其化学结构分别为:偏诺皂甙元3O-α-L-鼠李吡喃糖基(1→2)〔α-L-鼠李吡喃糖基(1→4))-β-D-葡萄吡喃糖甙(A);孕甾-5,16-二烯-3β-醇-20-酮,3β-O-α-L-鼠李吡喃糖基(1→2)〔α-L-鼠李吡喃糖基(1→4)〕-β-D-葡萄吡喃糖甙(B);孕甾-5,16-二烯-3β-醇-20-酮,3β-O-α-L-鼠李吡喃糖基(1→2)〔α-L-鼠李吡喃糖基(1→4)-α-L-鼠李吡喃糖基(1→4)〕-β-D-葡萄吡喃糖甙(C)。甙A、B和C在滇重楼根中尚未发现,甙C系首次从重楼属植物中获得,而甙A具有止血的活性。     

西南远志中一个新的三萜皂苷

从西南远志根中分离得到3个齐墩果酸型皂苷类化合物,根据理化性质和波谱数据鉴定其结构分别为3-O-β-D-葡萄糖基presenegenin 28-O-α-L-阿拉伯糖基-(1→3)-β-D-木糖基-(1→4)-[β-D-芹糖基-(1→3)]-α-L-鼠李糖基-(1→2)-[β-D-葡萄糖基-(1→3)]-[4-O-(E/Z)-3″,4″,5″-三甲氧基肉桂酰基]-β-D-岩藻糖基酯(1)、3-O-β-D-葡萄糖基presenegenin 28-O-β-D-木糖基-(1→4)-α-L-鼠李糖基-(1→2)-[α-L-鼠李糖基-(1→3)]-[4-O-(E/Z)-对甲氧基肉桂酰基]-β-D-岩藻糖基酯(2)和3-O-β-D-葡萄糖基presenegenin 28-O-α-L-阿拉伯糖基-(1→4)-β-D-木糖基-(1→4)-[β-D-芹糖基-(1→3)]-α-L-鼠李糖基-(1→2)-[4-O-(E/Z)-对甲氧基肉桂酰基]-[α-L-鼠李糖基-(1→3)]-β-D-岩藻糖基酯(3),其中化合物1为新化合物,化合物2和3首次从该植物中分离得到。     

产双功能褐藻胶裂解酶菌株的筛选与初步研究

目的：双功能褐藻胶裂解酶既能降解聚β-D-甘露糖醛酸,又能降解聚α-L-古罗糖醛酸,可以用一种酶来制备不同结构的褐藻胶寡糖。本文的目的是筛选能产生双功能褐藻胶裂解酶的菌株,对其产酶曲线和降解产物作初步研究。方法：利用唯一碳源培养基筛选产生褐藻胶裂解酶的菌株,通过16SrDNA序列比对进行菌种鉴定,通过在凝胶上检测褐藻胶裂解酶活性来判断发酵上清液中褐藻胶裂解酶的数量及分子量,利用薄层层析确定降解褐藻胶的终产物组成。结果：从褐藻上筛选到一株海洋细菌QY107,鉴定为弧菌属细菌。发酵120h时褐藻胶裂解酶产量为12．32U／mL,其发酵液上清中只含有一种褐藻胶裂解酶,分子量在28kDa左右,并且对聚β—D-甘露糖醛酸和聚α-L-古罗糖醛酸都能降解,降解褐藻胶的终产物主要为三糖。结论：本文筛选到一株弧菌QY107,其发酵液上清中只有一种双功能褐藻胶裂解酶,可用于大量制备褐藻胶三糖。推测该酶具有特殊的催化腔结构,对其结构与功能相互关系的研究可能会发现新的底物结合与催化机制。酶解制备褐藻胶寡糖因其环保高效而越来越受到人们的重视,因此该菌株能促进海洋寡糖类生物制品的开发,在医药、食品、农业、生物燃料等领域具有广阔的应用前景。     

微生物发酵-膜分离法制备褐藻胶寡糖及其产物分析

比较褐藻胶裂解酶产生菌Alteromonassp .在摇瓶和发酵罐培养过程中生物量、褐藻胶寡糖含量以及褐藻胶裂解酶活性的变化 ,根据其变化确立了通过微生物发酵 膜分离技术结合制备褐藻胶寡糖的条件 ,并对寡糖进行凝胶过滤色谱和薄层色谱分析。用组成为每升含酵母粉 5g、蛋白胨 10g、FeSO4 0 1g、褐藻酸钠 12g、NaCl 1 5g ,pH为7 5的培养基 ,在 2 8℃培养褐藻胶裂解酶产生菌 ,结果表明 ,发酵罐培养 30h ,发酵液寡糖含量达到最大。发酵液通过超滤 纳滤两级膜分离 ,得到褐藻胶寡糖 ,寡糖的回收率和脱盐率分别为 94 0 %和 93 3%。通过凝胶柱分离和TLC分析 ,得到 5个褐藻胶寡糖组分。     

Carbohydrate separation by hydrophilic interaction liquid chromatography on a 'click' maltose column

A general and efficient hydrophobic interaction liquid chromatography (HILIC) method has been developed for the separation of various kinds of carbohydrates, ranging from galactooligosaccharides, carrageenan oligosaccharides, sodium alginate, chitooligosaccharides to higher molecular weight fructooligosaccharides. Three types of oligosaccharides (acidic, basic, and neutral) were purified by semi-preparative HILIC. As a result, 18 oligosaccharides in high purity were obtained and their degrees of polymerization ranged from DP 2 to 7. Furthermore, oligosaccharides of plant origin were enriched and fractionated by solid-phase extraction (SPE) on a graphitized carbon cartridge. The oligosaccharide fractions that eluted from the cartridge were purified and then characterized by ESIMS and NMR spectroscopy. This strategy has led to the separation of a homogeneous raffinose family of oligosaccharides. Since the present method is universal, efficient and stable, separation and purification of various types of carbohydrates by HILIC seem to be possible.     

Enhancement of chrysogenin production in cultures of Penicillium chrysogenum by uronic acid oligosaccharides

Additions of 50 to 100 g of acid-hydrolysed alginate oligosaccharides ml^–1 and enzyme-hydrolysed pectin oligosaccharides to 24- to 48-h cultures of Penicillium chrysogenum, ATCC 9480, led to enhanced production of chrysogenin by over 30 to 40% in shaken flasks and bioreactors. Some of the oligosaccharides also promoted biomass formation but were not used as a carbon source.     

Isolation and characterization of alginate-degrading bacteria for disposal of seaweed wastes

Aims:  Isolation of novel alginate degrading bacteria for the disposal of seaweed waste in composting process.
Methods and Results:  Decomposition of alginate polymers was checked by the 3,5-dinitrosalicylic acid (DNS) method for reducing sugar, and absorbance at 235 nm for unsaturated sugar. A bacterium A7 was isolated from wakame compost and confirmed to belong to the genus Gracilibacillus by partial 16S rDNA analysis. The optimum condition for the growth of A7 in a medium containing 5 g l⁻¹ of sodium alginate is as follows: pH, 8·5–9·5; NaCl, 0·5 mol l⁻¹; temperature, 30°C and polypeptone as nutrient content, 2–5 g l⁻¹. In a laboratory-scale composting experiment, the alginate content in wakame compost decreased to 14·3% after 72 h of composting from an initial value of 36%, indicating the effectiveness of alginate decomposition of A7 in wakame composting.
Conclusions:  The bacterium A7 was found to be alginate lyase-producing in genus Gracilibacillus and effective in degrading alginate to oligosaccharides in wakame during composting process.
Significance and Impact of the Study:  Development of new methods for the disposal of marine wastes and production of functional products.     

系列海洋特征性寡糖抗紫外辐射构效关系研究

本文通过测定在紫外光胁迫下系列海洋特征性寡糖——褐藻寡糖、岩藻寡糖和壳寡糖分别对革兰氏阴性、阳性菌的抗辐射保护作用,并结合特征性寡糖的紫外吸收光谱及清除自由基能力初步探讨海洋活性寡糖抗紫外辐射作用机理及同分子量级海洋寡糖链间构效关系。结果表明三种活性寡糖均具有抗紫外线辐射作用,其效果与寡糖浓度呈正相关。三种海洋寡糖,抗辐射效果依次为褐藻寡糖>壳寡糖>岩藻糖。通过构效关系研究表明,抗辐射过程中特征性糖链的双键紫外吸收作用在细菌抗辐射保护过程中起主导作用,而特征性糖链的清除自由基能力具有辅助作用。     

The Surface Display of the Alginate Lyase on the Cells of Yarrowia lipolytica for Hydrolysis of Alginate

The alginate lyase structural gene (AlyVI gene) was amplified from plasmid pET24-ALYVI carrying the alginate lyase gene from the marine bacterium Vibrio sp. QY101 which is a pathogen of Laminaria sp. When the gene was cloned into the multiple cloning site of the surface display vector pINA1317-YlCWP110 and expressed in cells of Yarrowia lipolytica, the cells displaying the alginate lyase could form clear zone on the plate containing sodium alginate, indicating that they had high alginate lyase activity. The cells displaying alginate lyase can be used to hydrolyze poly-β-d-mannuronate (M) and poly-α-l-guluronate (G) and sodium alginate to produce different lengths of oligosaccharides (more than pentasaccharides). This is the first report that the yeast cells displaying alginate lyase were used to produce different lengths of oligosaccharides from alginate.     

褐藻胶裂解酶基因的克隆表达与酶学性质

随着大型褐藻生产燃料乙醇以及褐藻寡糖重大药用价值的发现,褐藻胶裂解酶成为国内外多个领域的研究重点。文中对解藻酸弧菌上与褐藻胶降解相关的5个基因分别进行克隆表达,通过SDS-PAGE和酶活性定量测定,发现该基因簇中的4个基因有降解褐藻胶活性。对酶活最高的rAlgV3进行了诱导条件的优化、酶蛋白纯化及酶性质研究,发现优化诱导条件后重组酶rAlgV3的酶活由2.34×10~4 U/L上升为1.68×10~5 U/L,比优化前提高了7.3倍;对酶性质进行表征发现该酶在4–70℃均有活性,最适反应温度为40℃,在4–20℃酶相对稳定;该酶在pH 6.5-9.0环境下均有较高的酶活,最适pH为8.0;pH稳定性好,在pH 4.5–9.5环境下可以稳定存在;适量的NaCl浓度和Fe~(2+)、Fe~(3+)等离子具有促进酶活的作用,SDS和Cu~(2+)离子可明显抑制酶活力。对该酶的底物特性的研究发现,该酶不仅可以降解褐藻胶中的Poly-M片段,也能降解Poly-G片段,具有广泛底物特性;其降解海藻酸钠主要释放二糖和三糖,是一种内切酶。该酶对于第三代燃料乙醇的发展及褐藻寡糖的生产具有重要作用。     

Co-immobilization of dextransucrase and dextranase in alginate

Dextransucrase from Leuconostoc mesenteroides and dextranase from Penicillium lilacinum were co-immobilized and used to produce isomaltooligosaccharides from sucrose. The enzymes were co-immobilized by encapsulating soluble dextransucrase and dextranase covalently attached to Eupergit C in alginate (beads, fibers, and capsules). The alginate capsule co-immobilization was done in the presence of soluble starch and resulted in a high immobilization yield (71%), and the enzymes retained their activities during 20 repeated batch reactions and for a month in storage at 4 °C. The presence of starch was essential for the stability of dextransucrase in alginate capsules. Furthermore, it is important that the dextranase be pre-immobilized prior to alginate capsule co-immobilization to prevent dextranase leakage and inactivation of dextransucrase. The co-immobilized enzymes formed oligosaccharides from sucrose, which can be used as prebiotics. In addition, the oligosaccharides that were produced after the addition of sucrose reacted with the alginate fiber-encapsulted dextransucrase, thus increasing the amount of prebiotics. Co-immobilization in alginate fiber and beads also resulted in high yields (70 and 64%), but enzymatic activities decreased by 74 and 99%, respectively, after a month in storage at 4 °C. The newly developed alginate capsule method for co-immobilization of dextransucrase and dextranase is simple yet effective and has the potential for industrial-scale production of isomaltooligosaccharides.     

Structural Studies of Alginic acid,using a bacterial poly-α-L-guluronate lyase

An extracellular poly-α-L-guluronate lyase from Klebsiella aerogenes degrades those blocks from alginate which contain both mannuronic and guluronic acid residues (poly-MG blocks) to a mixture of oligosaccharides. From an analysis of these products, it is concluded that poly-MG blocks do not have a strictly alternating sequence of the two uronic acid residues. Enzymic degradation of various samples of algal alginate to leave the poly-M blocks intact has shown that these blocks have a uniform chain-length, estimated at 24 residues.