苦荞籽粒芦丁降解酶的纯化、酶学性质及部分一级结构分析

芦丁降解酶催化芦丁降解为水溶性降低的槲皮素,是形成苦荞苦味的主要因素。研究以榆6-21苦荞籽粒粉为材料,通过硫酸铵分级沉淀、疏水层析、阳离子交换层析、凝胶过滤层析分离纯化芦丁降解酶,SDS-PAGE显示纯化的芦丁降解酶为分子量66 kDa的单一条带。芦丁降解酶的最适pH值为5.0,最适温度为50℃。对其催化特性研究表明,该酶的K_m值为0.27 mmol/L,V_(max)值为39.68 U/mg。Cu~(2+)、Zn~(2+)、Mn~(2+)和EDTA对其有不同程度的抑制作用。纯化的芦丁降解酶可耐受50%(V/V)的甲醇。Edman降解法测定其N端序列为TVSRSSFPDGFLFGL。MALDI-TOF-MS(基质辅助激光解吸电离飞行时间质谱)二级质谱获得了其15个内肽序列。该研究为从转录组数据中确定芦丁降解酶的候选基因及深入研究芦丁降解酶的生物学功能奠定了基础。     

陕北红枣中多糖的酶加水法提取工艺研究

以多糖含量和得率为指标,采用正交试验法对陕北红枣中多糖的提取与分离工艺进行优选。酶加水提取的最佳工艺为：①温度70℃、酶添加量3．0％、时间2h;②温度70℃、酶添加量2．0％、时间4h。分离的最佳工艺为：分级沉淀多糖时采用浓缩液：无水乙醇（v／v）1：3效果最好;当采用活性炭脱色时,活性炭用量为7g／L时,既达到了脱色的目的,多糖的损失率又较小。     

荞麦中总黄酮和芦丁含量的变化

苦荞、甜荞叶中总黄酮平均含量达5.3％,芦丁平均含量达4.9％,井在开花结实期达最高峰,后期下降;茎中的含量较低,平均值分别为1.0％和0.7％,生育期中无明显变化。叶、茎中黄酮成分以芦丁为主,占70％～90％。同一时期发育不同的生殖器官总黄酮及芦丁含量的大小顺序为蕾>花>胚乳未充实种子>成熟种子。生殖器官中黄酮成分与芦丁比重因荞麦种别而异。     

泥胡菜中芦丁含量分析

建立高效液相色谱法测定泥胡菜中芦丁的方法,优化芦丁的提取工艺。使用Diamonsil（TM）C18柱,流动相：乙腈-0．4％磷酸溶液（20：80）,检测波长：255nm,流速：1ml／min,柱温：30℃。样品用50％乙醇超声提取30min提取率最高为0．166％;芦丁在0．1～2．0μg范围内与峰面积呈良好的线性关系（r=0．9994）,平均加样回收率99．3％（RSD=0．8％,n=5）。该方法准确、快速、重现性好,可作为控制泥胡菜质量的有效测定方法。     

乙醇浸提法提取大枣芦丁及其抗氧化性的研究

以大枣为原料,采用乙醇浸提法提取芦丁,以芦丁得率为评价指标,通过正交试验优化影响大枣芦丁得率的若干因素:液料比、乙醇浓度、浸提时间、浸提温度,确定其最佳工艺条件为液料比20∶1(mL∶g)、乙醇浓度60%、浸提时间3.0 h、浸提温度70℃,此条件下大枣芦丁得率为2.16%;大枣芦丁粗提物中芦丁含量为8.57%。对芦丁进行体外抗氧化能力测定结果表明,大枣芦丁具有一定的清除DPPH自由基的能力,5μg/mL的大枣芦丁对DPPH自由基的清除率可达到58.80%,25μg/mL的芦丁对OH自由基的清除率达49.80%,本研究对芦丁资源的进一步开发利用提供参考。     

不同地区的紫苏种子中总黄酮的提取方法及其含量研究

为优化紫苏种子中总黄酮的提取方法,采用乙醇加热提取和超声波辅助提取,对提取液浓度、提取时间、提取温度开展了研究;并探究了不同地区的紫苏种子中总黄酮的含量,同时采用高效液相色谱法(HPLC)测定了不同地区的紫苏种子中芦丁和木犀草素的含量.试验结果表明:在用80％的乙醇溶液提取紫苏种子中的总黄酮时,温度控制在70℃,浸提1...     

苦荞突变体库构建与突变体中芦丁合成相关基因表达分析

苦荞是重要的小杂粮作物之一,营养物质丰富,是天然芦丁的重要来源。突破苦荞育种难题,创制苦荞新种质是目前研究的重要方面。本试验利用甲基磺酸乙酯(EMS)构建了黑丰1号苦荞突变体库,明确了当EMS浓度为1.2%时,诱变效果较好。通过对M1突变株表型观察统计,共获得叶色、叶型、株型、粒型变异单株102株,突变率为3.85%;高效液相色谱技术(HPLC)测定1000株M3材料,获得高芦丁含量突变株系2个和低芦丁突变体株系5个;qRT-PCR对芦丁含量突变体株系中芦丁代谢关键酶基因(CHS、F3H、4CL、FLS、UFGT)进行表达量分析,发现不同株系中上述基因的表达量与芦丁含量相关性不明显,但个别基因如FtFLS基因表达量在高芦丁含量突变体中达到对照的4.55倍。通过突变体的筛选丰富了苦荞基因资源,创新了苦荞新种质,也为苦荞芦丁代谢的分子基础研究提供了材料保证与技术支持。     

酶法提取银杏黄酮类化合物研究

本文研究了纤维素酶酶解法提取银杏总黄酮工艺。与传统的乙醇提取工艺相比,银杏总黄酮得率提高了18．92％。实验确定了最佳提取条件：酶浓度0．40mg／mL,酶作用时间120min,酶解温度50℃,酶解介质pH值为4．5,乙醇浓度70％,提取温度70℃。     

三株棒状杆菌降解环三次甲基三硝胺(RDX)的研究

从长期受RDX污染的土壤中分离到三株棒状杆菌,在含0．1％的适宜有机碳源的培养基中,1一3天内能使40一60mg／l的RDX降解90％以上。该细菌降解RDX的最适pH和温度分别为6．0--7．5和30℃。当培养基中加入氨态氮源时,它们对RDX的降解作用受到抑制。     

微生物转谷氨酰胺酶的纯化方法和酶学性质研究

由Streptoverticillium mobaTaense发酵生产的转谷氨酰胺酶经过除茵体、超滤浓缩、乙醇沉淀、干燥后得到粗酶产品,其活力回收率约70％。又经Superdex-75凝胶过滤和Source 30S阳离子交换两步纯化后得到纯酶,最终酶活力收率约37％。酶最适温度为50℃,在40℃以下稳定性良好;最适pH为6．0,pH4．0～8．0时比较稳定。离子强度对酶影响很小。     

凤尾蕨内生真菌的研究Ⅱ——1株产芦丁内生真菌初步研究

报道了从凤尾蕨属井栏边草(Pteris multifida)根状茎中分离出一内生真菌——菌株JJF006,对其形态特征进行了初步研究,并用TLC对该菌株培养物进行了分析。初步结果表明:该真菌液体培养基中含有芦丁成分。     

反相高效液相色谱法测定不同品种花椒中芦丁和槲皮素的含量

目的:建立RP-HPLC测定花椒中芦丁与槲皮素含量的方法,并对不同种花椒的中芦丁与槲皮素含量进行测定与比较。方法:Zorbax Eclipse C18色谱柱(150 mm×4.6 mm,5μm),流动相∶甲醇-0.4%磷酸(50∶50);流速1 mL/min;检测波长:360 nm;柱温25℃。结果:芦丁在0.25~5.0μg,r=0.999 9峰面积与质量浓度呈良好的线性关系;平均回收率为99.1%,RSD为4.3%(n=3)。槲皮素在0.25~0.5μg,r=0.999 9峰面积与质量浓度呈良好的线性关系;平均回收率为111.2%,RSD为5.1%(n=3)。结论:该方法可用于花椒中芦丁和槲皮素的测定。测定结果表明,韩城红花椒中芦丁含量最高,茂汉红花椒次之,四川青花椒较少,云南青花椒最低。槲皮素在韩城红花椒中含量较高,在其他三种花椒中差别不大。     

芸香甙热裂解研究

在 5 0 0℃和 770℃两种温度下 ,采用热解直接进样的方式 ,研究了烟草中主要的多酚类化合物———芸香甙的热裂解产物。分别鉴定出了 2 4个和 2 3个裂解化合物。通过比较和分析这两种温度下的裂解产物发现 ,芸香甙可以裂解产生大量具有香味的产物。不同温度下的热裂解产物中 ,其中有 16个热裂解产物是相同的 ,但它们的相对含量却不一样。     

一点红黄酮成分的分析及含量测定

对一点红中黄酮类成分进行了定性分析和定量测定,结果表明,在一点红中含有黄酮、黄酮醇、二氢黄酮、二氢黄酮醇等多种黄酮类化合物,并以芦丁为标样,用分光光度法测出总黄酮含量为4．02％。     

芦丁对胃蛋白酶部分性质的影响

采用紫外光谱和荧光光谱研究芦丁与胃蛋白酶的相互作用。实验结果表明,加入芦丁使胃蛋白酶的紫外吸收差谱迅速增强,特征荧光峰产生静态淬灭,并求得芦丁与胃蛋白酶作用的形成常数。     

Role of NF-κB/IL-1β Pathway and Caspase 3 in Mediating the Hepatoprotective Effect of Rutin against Paraquat-Induced Liver Toxicity in Male Rats

One of the most common bipyridinium herbicides that can lead to liver toxicity is paraquat. Rutin is a bioflavonoid with antioxidant, anti-inflammatory, anti-hepatotoxic, and antimicrobial properties. The effect of rutin on paraquat-induced liver toxicity was examined in this study. 48 male rats were divided into six groups: the control group was given a normal diet; the non-treated group was given paraquat; the positive control group was given paraquat, and silymarin and the treatment groups were given paraquat and rutin at doses of 25, 50, and 100 mg/kg. After fourteen days, the rats were anesthetized by xylazine-ketamine, and fasting blood samples were obtained from their hearts to measure alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT), malondialdehyde (MDA), creatinine, lipid profile, antioxidant capacity, and carbonyl protein. The liver tissue was removed to measure the levels of catalase (CAT), superoxide dismutase (SOD), total protein, vitamin C, plus NF-κB, IL1β, and caspase-3 gene expressions. Paraquat gavage in the untreated group (group 2) for 14 days in comparison with the control group induced a significant augmentation (p<0.05) in levels of lipid profile, AST, ALP, ALT, MDA, carbonyl protein, and also NF-κB, IL1β, Caspase3 expressions. Treatment with rutin reduced the factors as mentioned above. Paraquat poisoning induced a substantial decline (p<0.05) in HDL content, FRAP level, CAT, and SOD activity of the liver compared to the control group. However, rutin oral treatment led to a substantial increase (p<0.05) in the level of these factors compared to the paraquat-only treated group. Based on the findings of the present study, it was found that rutin can be significantly effective in improving hepatotoxicity caused by paraquat.     

Identification and characterization of a rhamnosyltransferase involved in rutin biosynthesis in Fagopyrum esculentum (common buckwheat)

Rutin, a 3-rutinosyl quercetin, is a representative flavonoid distributed in many plant species, and is highlighted for its therapeutic potential. In this study, we purified uridine diphosphate-rhamnose: quercetin 3-O-glucoside 6″-O-rhamnosyltransferase and isolated the corresponding cDNA (FeF3G6″RhaT) from seedlings of common buckwheat (Fagopyrum esculentum). The recombinant FeF3G6″RhaT enzyme expressed in Escherichia coli exhibited 6″-O-rhamnosylation activity against flavonol 3-O-glucoside and flavonol 3-O-galactoside as substrates, but showed only faint activity against flavonoid 7-O-glucosides. Tobacco cells expressing FeF3G6″RhaT converted the administered quercetin into rutin, suggesting that FeF3G6″RhaT can function as a rhamnosyltransferase in planta. Quantitative PCR analysis on several organs of common buckwheat revealed that accumulation of FeF3G6″RhaT began during the early developmental stages of rutin-accumulating organs, such as flowers, leaves, and cotyledons. These results suggest that FeF3G6″RhaT is involved in rutin biosynthesis in common buckwheat.     

Highly sensitive spectrofluorimetic determination of rutin based on its activated effect on a multi‐enzyme redox system

A highly sensitive and simple spectrofluorimetric method for the determination of rutin, based on its activated effect on a haemoglobin‐catalysed reaction, was developed. Under optimum conditions, the concentration of rutin was linear, with decreased fluorescence (ΔF) of the system under optimal experimental conditions. The calibration graph was linear in the range 1.0 × 10^–7–3.0 × 10^–5 mol/L, with a detection limit of 7.0 × 10^–8 mol/L. The relative standard deviation (RSD) was 3.26% for 11 determinations of 1.0 × 10^–5 mol/L. This method was used for the determination of rutin in pharmaceuticals with satisfactory results. Copyright © 2011 John Wiley & Sons, Ltd.