C18硅胶制备荔枝果皮原花青素低聚体的组成分析及单体分离

为更快速地从荔枝果皮中分离制备纯度更高的A-型原花青素低聚体,本研究在传统大孔树脂富集纯化的基础上,利用C18硅胶填料对制备方法进行了优化。通过组分分析和HPLC、LC-MS鉴定可知,新法制得的荔枝果皮原花青素低聚体(Litchi pericarp oligomeric procyanidins,LPOPC)总酚含量高达940±123 mg/g,且主要为(-)-表儿茶素、A-型原花青素二聚体和三聚体,相对含量分别占44.86%、36.02%和9.86%,平均聚合度约为1.62。因此,当再次通过Toyopearl HW-40s凝胶对提取物进行柱层析分级时,可得到纯度较高的已知A-型原花青素二聚体和三聚体。等辐射分析法(Isobologram)研究显示,该A-型二聚体和三聚体均可清除自由基,并呈现出协同抗氧化性。     

黄姑鱼(Nibe albiflora)肌肉乙酰胆碱酯酶的多分子形式和亚基组成

用Sepharosc 6B凝胶过滤和聚丙烯酰胺凝胶电泳方法观察了表面活性剂和NaCl对黄姑鱼(Nibea albiflora)肌肉乙酰胆碱酯酶分子形式的影响。在有2毫克/毫升TritonX-100存在时,酶以单体形式(Ⅰ)存在。去除TritonX-100即发生聚合,形成两种不同聚合度的多聚体(Ⅱ和Ⅲ)。NaCl的存在可以减缓而不能阻止酶的聚合作用。这时除单体形式外,还有酶的另一低聚形式(Ⅳ)、以及酶的高聚体(Ⅲ)存在。聚合的酶再经1%TritonX-100处理重新解离成单体形式。用Sepharose6B凝胶过滤方法测得酶的单体形式(Ⅰ)的分子半径为64(?)、低聚形式(Ⅳ)的分子半径为81(?)。用SDS-聚丙烯酰胺凝胶电泳方法测得单体酶分子是由相同的亚基组成,每个亚基的分子量为74,000。     

吸附层析法制备低聚原花青素

以AB 8大孔吸附树脂对葡萄籽原花青素粗提物进行了分级分离 ,所得乙酸乙酯洗脱产物总酚含量为 91% ,经高效液相色谱检测 ,酚类物质中单体约 2 8% ,低聚原花青素占 6 4 % ,多聚体仅为 8%。     

原花青素对亚硝化反应的抑制作用研究

研究了葡萄籽原花青素的最佳提取条件,及其对亚硝化反应的抑制作用。结果表明：原花青素的最佳提取条件为60％乙醇,料液比1：5,50℃水浴回流1h,其对亚硝胺合成的最大阻断率为91．2％,对亚硝酸钠的最大清除率为88．3％。     

端基法测定聚唾液酸平均聚合度

建立了端基法测定聚唾液酸平均聚合度的方法.采用间苯二酚比色法和丙二腈荧光法分别测定聚唾液酸中唾液酸总量和还原端唾液酸残基含量,两者之比即为平均聚合度.研究表明,在pH 9.5的硼酸缓冲液中,80℃水浴下聚唾液酸还原端唾液酸残基与丙二腈反应25 min后生成荧光物质,其荧光强度与还原端唾液酸残基含量呈线性正相关关系,线性范围在1～ 20 mg/L之间,变异系数和检出限分别为3.7％和0.36 mg/L.端基法测定大肠杆菌发酵液中聚唾液酸的平均聚合度为45.76,与高效液相凝胶色谱法比较,误差为3.2％.该法可以简便快速地测定发酵液中聚唾液酸的平均聚合度,有利于聚唾液酸生产过程分析及产品性能评估.     

低聚木糖的提取工艺及相对分子质量分布

通过单因素试验和正交试验研究低聚木糖的提取条件,使用凝胶树脂对低聚木糖粗提液进行分离,采用高效液相色谱法测定了低聚木糖的相对分子质量分布。低聚木糖的最佳提取条件:在底物质量浓度为100 g/L的情况下,加酶量1 000 U/g,酶解温度55℃,提取时间为4 h。在此条件下,提取的平均聚合度为3.12,高效液相色谱测定结果发现,低聚木糖主要是由木二糖、木三糖以及4～8个聚合度的糖组成。     

中国野生笃斯越橘花青素的初步分离和分析

采用反相高效液相色谱法和电喷雾质谱法对我国野生笃斯越橘中花青素组分进行了分离和分析．制备野生越橘花青素的粗提物,经大孔树脂柱层析得到花青素样品,其得率为0.72%．用香草醛法测得其总黄烷醇含量为95%．采用Alltima C18反相色谱柱(250 mm×4.6 mm,5 μm),以2 %甲酸水溶液(A)和2 %甲酸/甲醇溶液(B)作为流动相,分2个梯度对花青素组分进行洗脱,将其主要洗脱峰在电喷雾正离子模式下进行分析,根据质谱的准分子离子［M+H］(m/z)检测其聚合度．结果表明,我国野生越橘花青素的提取物被展开成8个主要的洗脱峰,以峰面积计算,主要洗脱峰总含量达85%．经质谱分析,判断为2个单体,3个二聚体,1个三聚体,2个四聚体．结果表明,我国野生笃斯越橘中含有较丰富的花青素,低聚体是其中的主要成分．本文首次报道了我国大兴安岭野生笃斯越橘花青素的组成情况,对于开展其后续的结构、功能、质量检测以及生产开发研究都具有一定的参考价值．     

超临界CO2萃取刺葡萄籽原花青素的工艺研究

刺葡萄(Vitis davidii Foex．)是属于葡萄属东亚种群的一个种,在湖南西部和南部的山区分布广泛,资源丰富,其果实酸甜、风味浓,但其果粒小,种子多,从种子中提取天然活性物质——原花青素的前景可能更为广阔。目前关于刺葡萄的研究报道少,为此本文以“紫秋”刺葡萄种子为原料,采用单因素试验和正交试验,探讨了超临界CO2萃取刺葡萄籽原花青素的工艺。研究结果表明,超临界CO2萃取刺葡萄籽原花青素的适宜工艺条件为:萃取压力30MPa,萃取温度55℃,料液比1:0．8,60％的乙醇为夹带剂,在此条件下,原花青素的产率为17．58 mg/100g,纯度可达89．7％。     

山葡萄籽中原花青素的提取

研究了提取溶剂、温度、时间、料液比4个因素对山葡萄籽中原花青素得率的影响。确定最佳提取工艺：以70％丙酮为提取荆,在60℃条件下,提取120min,料液比为1：7,原花青素的得率为2．31％     

链霉菌M-Z18膜蛋白ε-聚赖氨酸降解酶的分离纯化、酶学性质及应用

【目的】研究链霉菌Streptomyces sp.M-Z18ε-聚赖氨酸降解酶(Pld)的分离纯化及其生理生化特性,并利用该酶制备低聚合度ε-聚赖氨酸(ε-PL)。【方法】菌体细胞经超声破碎、NaSCN溶解和HiTrapTMButyl HP疏水层析制备到Pld,随后研究了其酶学性质、动力学和降解ε-PL过程,最后利用常量稀释法比较了不同聚合度范围ε-PL的最小抑菌浓度。【结果】从Streptomyces sp.M-Z18细胞膜上分离纯化到Pld,纯化倍数为80.4倍,回收率达到59.3%。以L-赖氨酰对硝基苯胺为底物,酶促反应的最适温度为37℃,最适pH为7.0,动力学常数Km为0.621 mmol/L,Vmax为701.16 nmol/min·mg;酶活在pH 7.0-10.0和50℃以下稳定。降解ε-PL实验发现,纯化到的Pld以内切方式降解ε-PL。抑菌实验表明,高聚合度ε-PL(30-35)对细菌的抑制效果较好,而低聚合度ε-PL(8-20)更有利于抑制酵母菌的生长,各种聚合度ε-PL对霉菌的生长抑制均较差。【结论】从ε-PL产生菌中分离纯化到内切型ε-PL降解酶,发现不同聚合度范围ε-PL对微生物的抑制能力存在显著差异。     

Polymer masked-unmasked protein therapy. 1. Bioresponsive dextrin-trypsin and -melanocyte stimulating hormone conjugates designed for alpha-amylase activation

Polymer-protein conjugation, particularly PEGylation, is well-established as a means of increasing circulation time, reducing antigenicity, and improving the stability of protein therapeutics. However, PEG has limitations including lack of polymer biodegradability, and conjugation can diminish or modify protein activity. The aim of this study was to explore a novel approach for polymer-protein modification called polymer-masking-unmasking-protein therapy (PUMPT), the hypothesis being that conjugation of a biodegradable polymer to a protein would protect it and mask activity in transit, while enabling controlled reinstatement of activity at the target site by triggered degradation of the polymeric component. To test this hypothesis, dextrin (alpha-1,4 polyglucose, a natural polymer degraded by alpha-amylase) was conjugated to trypsin as a model enzyme or to melanocyte stimulating hormone (MSH) as a model receptor-binding ligand. The effect of dextrin molecular weight (7700, and 47200 g/mol) and degree of succinoylation (9-32 mol %) on its ability to mask/unmask trypsin activity was assessed using N-benzoyl-L-arginine-p-nitroanilide (L-BAPNA). Dextrin conjugation reduced enzyme activity by 34-69% depending on the molecular weight and degree of succinoylation of dextrin. However, incubation with alpha-amylase led to reinstatement of activity to a maximum of 92-115%. The highest molecular dextrin (26 mol % succinoylation) gave optimum trypsin masking-unmasking. This intermediate was used to synthesize a dextrin-MSH conjugate (dextrin Mw = 47200 g/mol; MSH content 37 wt %), and its biological activity (+/-alpha-amylase) was assessed by measuring melanin production by murine melanoma (B16F10) cells. Conjugation reduced melanin production to 11%, but addition of alpha-amylase was able to restore activity to 33% of the control value. These were the first studies to confirm the potential of PUMPT for further application to clinically important protein therapeutics. The choice of masking polymer, activation mechanism, and the rate of unmasking can be tailored to therapeutic application.     

Content and mean polymerization degree of procyanidins in extracts obtained from clear and cloudy apple juices

The polyphenol profile of apples and that of technologically differently treated apple juices has already been studied thoroughly; nevertheless, the content of polymeric procyanidins has not received much attention up to date. Therefore, procyanidins in extracts made from six blended apple juices and two authentic clear as well as cloudy apple juices (Malus domestica cv. Bohnapfel and Bittenfelder) were investigated. Our determinations revealed significant differences in the total procyanidin content between apple juice extracts obtained from clear and the corresponding cloudy juices under study. Depending on the apple cultivars used average amounts of total procyanidin content determined in the extracts made from clear and cloudy juices ranged from 28.4 +/- 4.4% to 49.0 +/- 5.7% and from 48.3 +/- 0.3% to 60.6 +/- 0.3%, respectively. As the mean degree of polymerization (DPm) is supposed to have an influence on bioavailability and toxicity on different cells lines used in in vitro systems, the average degree of polymerization of the juices under examination were determined. Depending on the cultivar used and the technology of juice processing the DPm ranged between 3.0 and 13.4.     

葡萄籽中原花青素提取方法优化处理

溶剂提取法是提取葡萄籽原花青素的常用方法 ,然而 ,在不同提取条件下 ,提取效果并不一致。利用水、甲醇、乙醇、丙酮及它们的水溶液提取葡萄籽中的原花青素 ,而后用铁盐催化比色法测原花青素含量 ,考察了提取剂的浓度、粉碎度对提取的影响。在优化处理的基础上 ,获得了有效的提取条件 :葡萄籽粉碎度 10 0目 ,提取剂 70 %甲醇水溶液。     

Formulation of Controlled-Release Baclofen Matrix Tablets II: Influence of Some Hydrophobic Excipients on the Release Rate and In Vitro Evaluation

The aim of this study was to investigate the influence of polymer level and type of some hydrophobic polymers, including hydrogenated castor oil (HCO); Eudragit RS100 (E-RS100); Eudragit L100 (E-L100), and some fillers namely mannitol [soluble filler], Dibasic calcium phosphate dihydrate (Emcompress) and anhydrous dibasic calcium phosphate [insoluble fillers] on the release rate and mechanism of baclofen from matrix tablets prepared by a hot-melt granulation process (wax tablets) and wet granulation process (E-RS100 and E-L100 tablets). Statistically significant differences were found among the drug release profile from different classes of polymeric matrices. Higher polymeric content (40%) in the matrix decreased the release rate of drug because of increased tortuosity and decreased porosity. At lower polymeric level (20%), the rate and extent of drug release was elevated. HCO was found to cause the strongest retardation of drug. On the other hand, replacement of Emcompress or anhydrous dibasic calcium phosphate for mannitol significantly retarded the release rate of baclofen, except for E-L100 (pH-dependent polymer). Emcompress surface alkalinity and in-situ increase in pH of the matrix microenvironment enhanced the dissolution and erosion of these matrix tablets. The release kinetics was found to be governed by the type and content of the excipients (polymer or filler). The prepared tablets showed no significant change in drug release rate when stored at ambient room conditions for 6 months.     

Influence of flavan-3-ols and procyanidins on UVC-mediated formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in isolated DNA

The flavan-3-ols (-)-epicatechin (epicatechin) and (+)-catechin (catechin) and their related oligomers (procyanidins) isolated from cocoa were assayed for their capacity to inhibit the UVC-mediated formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo(8)dG) in calf thymus DNA. The above-mentioned compounds inhibited oxo(8)dG production in a concentration- and time-dependent manner. After 30 min of irradiation (30 kJ/m(2)), 0.1, 1.0, 10, and 100 microM epicatechin inhibited oxo(8)dG formation by 20, 36, 64, and 74%, respectively. For the same dose of UVC, 0.1, 1.0, 10, and 100 microM catechin inhibited oxo(8)dG formation by 1, 23, 50, and 70%, respectively. Epicatechin was more efficient than catechin with respect to inhibiting oxo(8)dG formation (IC(50) 1.7 +/- 0.7 vs 4.0 +/- 0.7 microM). Monomer, tetramer, and hexamer fractions were equally effective in inhibiting oxo(8)dG formation when assayed at 10 microM monomer equivalent concentration. At similar concentrations (1-50 microM), the inhibition of the UVC-mediated oxo(8)dG formation by flavan-3-ols and procyanidins was in the range of that of alpha-tocopherol, Trolox, ascorbate, and glutathione. These results support the concept that flavan-3-ols and their related procyanidins can protect DNA from oxidation at concentrations that can be physiologically relevant. Both epimerism and degree of oligomerization are important determinants of the antioxidant activity of flavan-3-ols and procyanidins.     

不同磷水平配施生物炭对土壤磷有效性

生物炭在提高土壤磷素有效性及促进作物生长方面具有显著作用,但其效果因土壤类型不同存在较大差异。试验以赤红壤(pH 4.91)和褐土(pH 7.24)为供试土壤,设置3种磷肥水平(0、30、90 kg P·hm^-2,分别以不施磷、低磷、高磷表示)配施稻秆生物炭(0、4%)的大豆盆栽试验,研究了不同磷水平下配施生物炭对土壤磷有效性、磷酸单酯酶活性和植株磷吸收的影响。结果表明: 不同磷水平配施生物炭显著提高了两种土壤的速效磷和全磷含量,且低磷水平添加生物炭处理速效磷增幅最大,在赤红壤和褐土的增幅分别为192.6%和237.1%。与低磷相比,赤红壤中低磷配施生物炭处理的碱性磷酸单酯酶活性显著增加78.9%,活性有机磷含量降低39.3%,同时显著促进了植株生长与磷吸收;生物炭添加显著降低了褐土活性有机磷含量,但不同处理对土壤磷酸单酯酶活性和植株生长无显著影响。土壤活性有机磷含量与速效磷含量均呈显著负相关。综上,生物炭对土壤磷有效性的作用因土壤类型和磷肥水平差异而不同,其在赤红壤上对植株生长和磷吸收的促进效应强于褐土,且在低磷条件下效果更佳。本研究为生物炭在减施磷肥和促进大豆磷吸收,特别是在赤红壤上的应用提供了科学依据。     

Correlation between Plant Growth Regulator Release Rate and Bioactivity for the Series of Newly Synthesized Phytoactive Polymers

Phytoactive polymers are high molecular weight systems in which a plant growth regulator (PGR) unit is attached to the polymeric chain by a hydrolyzable chemical bond. The release rate of the PGR is linked to the biological activity of the phytoactive polymer and can be controlled by properties inherent in the whole macromolecular system. In this study the correlation of biological activity and plant growth regulator hydrolytic release rate was investigated for the series of newly synthesized 2,4-dichlorophenoxyacetic acid (2,4-D) polymeric esters. The polymers synthesized differ in their molecular weight, side group structure, and 2,4-D residue content. The influence of these polymer characteristics on the 2,4-D hydrolytic release was investigated, and it was demonstrated that hydrolysis rate substantially depends on the polymer molecular weight, side group structure, and 2,4-D residue content. It was also demonstrated that phytoactive polymer bioactivity depends on the hydrolysis rate of the polymers, and in dependence of this parameter can provide stimulating or inhibiting activity. Biological activity was illustrated by the elongation of wheat and barley coleoptiles.     

Changes in grape seed polyphenols during fruit ripening

The quantity and characterization of extracted flavan-3-ol monomers and procyanidins was determined in seeds from Vitis vinifera cv. Cabernet Sauvignon berries, over the course of ripening and at different levels of vine water status. The per berry extractive yield of all polyphenols decreased with maturity, and followed second-order kinetics. The flavan-3-ol monomers decreased most rapidly, followed by the procyanidin extension units and finally, the terminal units. The relative proportion of procyanidin extension units did not vary with maturity. During fruit ripening, the mean degree of polymerization of extracted procyanidins is unchanged when analyzed intact by HPLC, but decreases by thiolytic degradation. The proportion of extracted procyanidins resistant to acid catalyzed thiolysis increased with maturity. Changes in vine water status affected polyphenol amounts, indicating that cultural practices can be used to influence composition. Oxidation of the seed polyphenols during fruit ripening, could explain these observations.     

The formation of catechin and procyanidins in cell suspension cultures ofCryptomeria japonica

Cell suspension cultures, as well as green leaves, ofCryptomeria japonica contained catechin, epicatechin, procyanidins B-1, B-3 and B-4, and polymeric procyanidins. Those compounds in the cell culture were found to increase, midway through the logarithmic phase, but the polymerization of procyanidins seems to proceed in the stationary phase. The dosage of 0.3 mMl-2-aminooxy-3-phenylpropionic acid (l-AOPP), an inhibitor of phenylalanine ammonia-lyase (PAL), to the cells caused inhibition of the flavan formation to a large extent without significant reduction of growth rate, as well as a large increase in the phenylalanine content of the cells. PAL activity in the cell cultures increased, immediately after transfer to a fresh medium, showed its maximum (a first peak) during 15 hr and a second small peak of the activity in the midst of the logarithmic phase. 0.3 mMl-AOPP inhibited remarkably a first peak of PAL activity, but a second peak was nearly unaffected. 2 mMl-AOPP inhibited the PAL activity completely.     

The anti-translocation and anti-inflammatory effect of cinnamon oil in mice with TNBS induced colitis

The bacterial translocation induced by colitis may cause the organ failure and sepsis. Therefore, it is necessary to find new possibilities for prevention and therapy of this problem. The purpose of this study was to examine Escherichia coli anti-translocation activity of cinnamon oil and its ability to reduce colonic damage in mice with TNBS (2,4,6-trinitrobenzenesulfonic acid) induced colitis. Mice received cinnamon essential oil in four various concentrations (0.5%, 0.25%, 0.125% and 0.063%) in the powdery commercial rodent diet, starting 21 days before induction of TNBS colitis. The colonic damage was assessed using the colon macroscopic scoring system (Wallace score). E. coli translocation to the mesenteric lymphatic nodules was evaluated by serial dilutions method for counting bacteria. Bacterial translocation was significantly reduced in first and third group (15.2% or 42.8% in cinnamon oil groups versus 100% in TNBS group). Cinnamon oil was effective also against the colonic damage in all cinnamon oil groups (macroscopically scores of grade 9 in TNBS group versus 5.25, 5.63, 5.13 and 3.25 in cinnamon oil groups). Our results confirmed that dietary administration of cinnamon oil could possess potential therapeutic effects on bacterial translocation and intestinal wall injury in colitis.