槲皮素对胰蛋白酶活性的影响

通过测定槲皮素对胰蛋白酶催化活性、催化反应动力学以及内源荧光光谱的影响,对槲皮素和胰蛋白酶相互作用特性进行研究.结果表明:槲皮素对胰蛋白酶催化活性有明显的抑制作用,当槲皮素与胰蛋白酶的摩尔比为44∶1,在37℃反应10min,抑制率达到32.5％;反应时间对两者的作用影响并不明显;槲皮素对胰蛋白酶催化活性的抑制作用属于可逆的竞争性抑制;槲皮素可使胰蛋白酶的内源荧光发生猝灭现象,猝灭常数Kq是4.7415×1012 (mol/L)1·S-1,猝灭类型为静态猝灭,结合位点数N为0.9206.     

槲皮素与β-乳球蛋白B的结合机制及其溶解性能

为解决槲皮素水溶性差的问题,采用β-乳球蛋白B作为载体,基于多光谱、等温滴定量热仪和分子模拟等实验手段,探究槲皮素和β-乳球蛋白B的结合机制,以及结合后β-乳球蛋白B的二级结构和槲皮素溶解度的变化。结果表明：槲皮素可与β-乳球蛋白B结合,该结合过程中氢键和范德华力起主要作用;三维荧光、同步荧光以及紫外-可见吸收光谱表明,槲皮素的结合使β-乳球蛋白B的二级结构发生了改变;圆二色谱和傅里叶变换红外光谱结果表明,槲皮素的结合诱导该蛋白中部分α-螺旋转变为β-折叠;分子模拟结果表明,槲皮素在β-乳球蛋白B上的结合位点位于一个由α-螺旋与β-转角所形成的空穴中,在该结合位点中有8 个氨基酸残基参与了与槲皮素的结合,其中第125位的苏氨酸残基提供了较强的氢键,其余残基则提供了范德华力;基于高效液相色谱检测发现在与β-乳球蛋白B结合后,槲皮素的溶解度增大至原来的1 844 倍左右。综上所述,以β-乳球蛋白B为载体结合的槲皮素水溶性显著提高。     

Interaction between four flavonoids and trypsin: effect on the characteristics of trypsin and antioxidant activity of flavonoids

Interaction of flavonoids and enzyme may affect characteristics and physiological activities of both components. In this study, the effects of the interaction between four flavonoids (quercetin, luteolin, kaempferol and apigenin) and trypsin were examined. At the concentration of 2.7 mm , inhibition of trypsin (1.6 U mL^?1) by quercetin, luteolin, kaempferol and apigenin was 46.4%, 32.6%, 26.8% and 17.7%, respectively. In the presence of trypsin, DPPH, ABTS and hydroxyl radical scavenging activities of flavonoids were obviously inhibited. Addition of flavonoids led to fluorescence quenching of trypsin. The decreasing order of binding force between flavonoids and trypsin was quercetin, luteolin, kaempferol and apigenin. It is concluded that the interaction between flavonoids and trypsin depends on the number and position of hydroxyl group of flavonoids.     

槲皮素对酪氨酸酶的抑制作用与分子机理

本文以L-多巴为底物,采用酶抑制动力学法研究了槲皮素对酪氨酸酶的抑制作用大小及类型,并采用荧光光谱技术分析其与酪氨酸酶的猝灭作用类型、结合位点、作用力类型。在此基础上,进一步利用柔性分子对接技术分析槲皮素对酪氨酸酶的抑制机理。结果表明,槲皮素对酪氨酸酶具有抑制作用,抑制常数KI为36 m M,以竞争性抑制剂形式抑制酪氨酸酶活性,是一种可逆性抑制剂;槲皮素以1:1比例通过氢键和疏水作用力结合于酪氨酸酶活性中心,且对酪氨酸酶的荧光产生静态淬灭作用,具有氢键及疏水作用力;分子对接结果验证了以上实验结论:槲皮素占据了酪氨酸酶活性中心,且与活性中心部位的Asn260和Gly62残基形成了强烈的氢键作用,同时伴有疏水作用共同稳定复合物的结构。     

NaCl浓度对麦醇溶蛋白与槲皮素相互作用的影响

利用荧光光谱、紫外光谱、傅里叶变换红外光谱法,研究不同NaC1浓度下麦醇溶蛋白(gliadin,G)与槲皮素(quercetin,Q)的相互作用.荧光光谱分析结果表明:不同NaC1浓度下,Q导致G荧光猝灭现象的发生,且随NaCl浓度的增大,荧光强度伴随明显蓝移现象(10 nm左右);当Q浓度为50 μmol/L时,荧光...     

Preferential binding of SBD from Arabidopsis thaliana SSIII to polysaccharides: Study of amino acid residues involved

SS III (SSIII) has been reported to play a regulatory role in the synthesis of transient starch. SSIII from Arabidopsis thaliana contains 1025 amino acid residues and has an N‐terminal transit peptide for chloroplast localization followed by three in tandem starch‐binding domains (SBDs D1, D2, and D3, residues 22‐591). Its C‐terminal catalytic domain (residues 592–1025) is similar to bacterial glycogen synthase. Binding studies to raw starch and its individual components, AM or AP show that the SBD region binds preferentially to AM, and that the D1 domain is mainly responsible for this selective binding. The D2 domain contains two binding sites which include amino acid residues Y394 (binding site 1) and W366 (binding site 2) acting cooperatively with the D1 domain in the binding process while G335 and W340 have a minor role. In addition, mutations in these residues also affect the kinetic parameters for the polysaccharide substrate of SSIII.     

The influence of flavonoids on the binding of pantoprazole to bovine serum albumin by spectroscopic methods: With the viewpoint of food/drug interference

The interaction of pantoprazole to bovine serum albumin (BSA) has been investigated with and without four popular 5,7,3',4'-hydroxy-substituted flavonoids, quercetin, luteolin, taxifolin and (+)-catechin. The presence of flavonoids decreased binding constants of pantoprazole with BSA from 39.7% to 93.8%, which depended on flavonoid structures. Analysis of infrared spectroscopy (IR) and circular dichroism (CD) showed the binding of pantoprazole to BSA caused apparent change in secondary structure of BSA. The calculated values of spatial-distance indicated the existence of quercetin, luteolin and taxifolin may compete with pantoprazole binding to BSA, while the existence of (+)-catechin possibly formed ternary pantoprazole-BSA-(+)-catechin complex. However, all the fluorescence quenching was initiated by static quenching procedure irrespective of the absence or presence of flavonoids, while van der Waals force and hydrogen bonds played major roles for pantoprazole-BSA association. All above results may have relevant consequence in rationalising the interferences of common food to gastric ulcer treatments.     

Quercetin dietary supplementation of fattening lambs at 0.2% rate reduces discolouration and microbial growth in meat during refrigerated storage

Thirty-two Merino lambs fed barley straw and a concentrate formulated either with palm oil (CTRL group) plus quercetin (QCT group) or flaxseed (FS group) plus quercetin (FS-QCT group) were used to assess the effects of this flavonoid on meat quality attributes. The animals were slaughtered after being fed for at least 5 weeks with the experimental diets. Chemical composition of longissimus thoracis (LT) muscle was not different among treatments. The longissimus lumborum (LL) samples of QCT and FS-QCT groups revealed lower discolouration (hue angle) when compared to the CTRL and FS lambs (P < 0.05), whereas extract release volume (ERV) and microbiological data jointly suggest that flaxseed and quercetin may reduce the growth of microbial populations responsible for meat spoilage in quadriceps femoris (QF).     

In vivo Mutational Analysis of Highly Conserved Amino Acid Residues of the Small Subunit Cpa1p of the Carbamylphosphate Synthetase of Saccharomyces cerevisiae

The role of selected amino acid residues located in the putative catalytic domain and of two conserved histidine residues within the small subunit of the carbamylphosphate synthetase (CPS) specific to the arginine biosynthesis pathway of the yeast Saccharomyces cerevisiae was studied using site-directed mutagenesis to change all residues to aspartic acid. Carbamylphosphate synthesis catalysed by modified CPS was tested in vivo. The C264D, H307D and H349D mutants were unable to grow on minimal medium, indicating the importance of these three residues for efficient CPS activity, whereas, four other mutated residues located in the catalytic site (including a proline residue) do not affect the growth rate. These results in comparison to those obtained with the CPS of Escherichia coli, implicate residues Cys 264 and His 349 in the glutaminase catalytic activity, and His 307 in the binding of glutamine to the active site. Using these three defective mutants, we investigated the in vivo utilization of ammonia by CPS. C264D and H307D mutants are able to use ammonia as a substrate when provided in sufficiently high concentrations (up to 200 mm). The H349D mutant, however, did not grow even at ammonium sulfate concentrations above 400 mm, suggesting that this substitution is critical to NH₃-dependent CPS activity although the ammonia binding site is presumably located within the large subunit of the enzyme. © 1997 by John Wiley & Sons, Ltd.     

Preparation and properties of rutin-hydrolyzing enzyme from tartary buckwheat seeds

A rutin hydrolyzing enzyme (RHE) was isolated from Fagopyrum tataricum Moench seeds by using ammonium sulphate fractionation, anion exchange and size exclusion chromatography. The purified RHE has an apparent molecular weight of about 70 kDa determined by SDS-PAGE, with an isoelectric point (pI) (determined by isoelectric focusing) of 6.7. RHE has a specific catalytic activity toward rutin when incubated together with rutin at 37 °C for 30 min in the presence of 20% ethanol, and its K_m value for rutin is 1.04 × 10⁻³ M. The RHE catalytic product analyzed by HPLC displayed high similarity with quercetin and this is confirmed by ¹H NMR spectroscopy and LC-ESI-MS/MS, suggesting that the RHE hydrolysis product is quercetin. These results suggest that the RHE from tartary buckwheat seeds is a specific rutin-hydrolyzing enzyme, providing a new enzymatic preparation method for quercetin.     

Determining the binding affinities of phenolic compounds to proteins by quenching of the intrinsic tryptophan fluorescence

The noncovalent binding of selected phenolic compounds (chlorogenic-, ferulic-, gallic acid, quercetin, rutin, and isoquercetin) to proteins (HSA, BSA, soy glycinin, and lysozyme) was studied by an indirect method applying the quenching of intrinsic tryptophan fluorescence. From the data obtained, the binding constants were calculated by nonlinear regression (one site binding; y = Bx/k + x). It has been reported that tannins inhibit human salivary amylase and that these complexes may reduce the development of cariogenic plaques. Further, amylase contains two tryptophan residues in its active site. Therefore, in a second part of the study involving 31 human subjects, evidence was sought for noncovalent interactions between the phenols of green tea and saliva proteins as measured by the fluorescence intensity. Amylase activity was determined before and after the addition of green tea to saliva of 31 subjects. Forty percent of the subjects showed an increase in amylase activity contrary to studies reporting only a decrease in activity. The interactions of tannin with amylase result in a decrease of its activity. It still remains to be elucidated why amylase does not react uniformly under conditions of applying green tea to saliva. Further, in terms of using phenols as caries inhibitors this finding should be of importance.     

Improved aqueous solubility,bioaccessibility and cellular uptake of quercetin following pH-driven encapsulation in whey protein isolate

The purpose of this study was to increase the water solubility and potential bioavailability of quercetin by encapsulation in whey protein isolate (WPI) based on a green, efficient pH-driven method. According to the results, the water solubility of quercetin increased by 346.9: times after loading into WPI nanoparticles. When the initial quercetin concentration was 0.25 mg mL⁻¹ and WPI was 2% w/v, the encapsulation efficiency reached 94.1%, the Z-average diameter was 36.63 nm, and the zeta potential was −36.4 mV at pH 7.0. The fluorescence spectroscopy assay suggested the molecular complexation of quercetin and WPI at pH 12.0. X-ray diffraction assay indicated the enclosure of amorphous quercetin in WPI. Correspondingly, the bioaccessibility increased from 2.76% to 31.23% and the Caco-2 cell monolayer uptake increased from 0% to 2.12% after nanoencapsulation. This work confirmed that the pH-driven method is an effective approach to prepare WPI–quercetin nanocapsules to improve physical and potentially biological properties of quercetin.     

苦味酸与牛血清蛋白相互作用的光谱研究

运用荧光和紫外光谱法并结合分子模拟技术研究了人体生理条件下苦味酸(Picric acid,简称PA)与牛血清白蛋白(BSA)的结合作用。实验结果表明,苦味酸对BSA的荧光猝灭作用较强,该猝灭属于生成复合物的静态猝灭。根据实验结果求得了不同温度下苦味酸与牛血清白蛋白结合作用的结合位点数和结合常数。根据van’t Hoff方程计算出的热力学参数,确定它们之间的相互作用力由以氢键和范德华力为主转变为以疏水作用力为主。根据Foster非辐射能量转移理论,求得苦味酸在距色氨酸残基距离3.14nm的位置与BSA结合。三种探针竞争标记药物实验表明,苦味酸主要结合在BSA上site I位点,分子模拟证实了该结合位点。同步荧光光谱结果表明苦味酸的加入使BSA的构象发生了变化。      

槲皮素、芦丁与大豆分离蛋白非共价作用机制及其功能性和消化性研究

本文分析了槲皮素-大豆分离蛋白与芦丁-大豆分离蛋白复合物功能性(溶解性、乳化性、凝胶性)和消化性的变化,并利用紫外可见光谱法、荧光光谱法研究互作机理,解析了其荧光淬灭类型、结合位点数以及热力学参数。结果发现,两种黄酮均可提高SPI的功能性质,当槲皮素与芦丁添加量分别为8%时,SPI凝胶硬度可分别提高23.23%及187.18%;随着槲皮素添加量的增加,SPI 的 EAI、 ESI 和溶解性先增加后趋于平缓,添加量为6%和4%时,乳化活性和溶解性分别达到最高,与SPI相比,分别提高 20.84%和 10.06%;随着芦丁添加量的增加,SPI 的 EAI、 ESI 和溶解性先增加后略有下降,在添加量为4%和6%时,乳化活性和溶解性分别达到最高,与SPI相比,分别提高26.17%和19.27%。此外,槲皮素、芦丁分别与SPI相互作用后还可提高蛋白的生物利用度,进一步研究两种黄酮多酚与SPI互作机制表明,两种互作复合物的荧光光谱均发生蓝移现象,槲皮素、芦丁与SPI自发结合,并主要通过氢键和范德华力方式作用,其中槲皮素、芦丁与SPI互作机制分别为动态淬灭及静态淬灭。     

Flaked Sinew Addition to Low-fat Cooked Salami

Flaked sinew (FS) was added at three levels (7.5, 15, and 22.5%) to low-fat, 20% added water (AW) salamis. A 1.27 cm Comitrol machine head was used to produce large (flaked once) and small (flaked twice) particle sizes. Treatments were preblended (P) and non-preblended (N). P and N controls contained 10% and 20% fat, respectively. Addition of FS decreased (P < 0.05) moisture compared to controls. Yields decreased (P < 0.05) as FS increased regardless of particle size; however, FS addition minimized purge. Lightness increased (P < 0.05) as FS increased in N treatments compared to the low-fat control. Break force/cm² increased (P < 0.05) in P controls compared to N controls. Large FS particle size N treatments had higher (P < 0.05) peak force values than those with small particle size. FS treatments were comparable to high-fat controls in palatability traits.     

黑曲霉降解黄曲霉毒素B1的研究及转录组分析

为探究黑曲霉FS10对AFB_1的降解机制,使用黑曲霉FS10的不同组分(菌悬液、发酵液、孢子、菌丝体)对AFB_1进行降解,并研究了AFB_1刺激对黑曲霉FS10降解效果的影响;利用扫描电子显微镜观察降解过程中黑曲霉菌丝体的形态变化;用转录组学技术探究AFB_1可能的降解机理。结果表明:黑曲霉FS10能有效降解AFB_1,72 h时菌悬液的对AFB_1脱除率高达98.65%;黑曲霉FS10孢子对AFB_1无明显脱除作用,但菌丝体对AFB_1有一定的吸附能力,发酵液对AFB_1有明显脱除效果;经过AFB_1诱导刺激后黑曲霉FS10降解效果有明显提升,表明AFB_1处理能显著提升黑曲霉FS10对AFB_1的降解能力。微观结构分析表明AFB_1处理在一定程度上影响黑曲霉FS10的形态,但随着时间的延长这种影响逐渐减小。此外,转录组学分析表明AFB_1处理降低了一些能量代谢基因的水平,这可能是黑曲霉FS10的一种自我保护机制,同时蛋氨酸的合成基因上调,推测AFB_1的降解可能与蛋氨酸的合成有关。     

Isolation and characterization of the carbon catabolite‐derepressing protein kinase Snf1 from the stress tolerant yeast Torulaspora delbrueckii

We cloned a genomic DNA fragment of the yeast Torulaspora delbrueckii by complementation of a Saccharomyces cerevisiae snf1Δ mutant strain. DNA sequence analysis revealed that the fragment contained a complete open reading frame (ORF), which shares a high similarity with the S. cerevisiae energy sensor protein kinase Snf1. The cloned TdSNF1 gene was able to restore growth of the S. cerevisiae snf1Δ mutant strain on media containing nonfermentable carbon sources. Furthermore, cells of the Tdsnf1Δ mutant were unable to proliferate under nonfermenting conditions. Finally, protein domain analysis showed that TdSnf1p contains a typical catalytic protein kinase domain (positions 41–293), which is also present in other Snf1p homologues. Within this region we identified a protein kinase ATP‐binding region (positions 48–71) and a consensus Ser/Thr protein kinase active site (positions 160–172). The GenBank Accession No. for the sequenced DNA fragment is HM131845. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of total phenols in tea infusions,tomato and apple juice by terbium sensitized fluorescence method as an alternative approach to the Folin–Ciocalteu spectrophotometric method

A fast screening of total phenols in tea infusions, tomato and apple juice samples using terbium sensitized fluorescence is described. The proposed method is based on the fluorescence sensitization of terbium (Tb³⁺) by complexation with flavonols (quercein as a reference standard) (at pH 7.0), which fluoresces intensely with an emission maximum at 545 nm when excited at 310 nm. Quercetin and terbium cations (at pH 7.0) form a stable complex and the resulted emission at 545 nm can be used for the determination of the total phenols concentration expressed in terms of “quercetin equivalent”. Based on the obtained results, a sensitive, simple and rapid spectrofluorimetric method was developed for the determination of total phenols. In the optimum conditions, the calibration graph was linear from 0.01 to 2 μg mL⁻¹, with the limit of detection of 0.002 μg mL⁻¹. The relative standard deviation values were in the range of 0.75–2.3%. The total concentrations of quercetin equivalent in five tested samples were found in the range of 6.6–27.9 μg mL⁻¹ and the results compare favorably with those obtained by spectrophotometric method (r = 0.999).     

Non-toxic Salvia sclareoides Brot. extracts as a source of functional food ingredients: Phenolic profile,antioxidant activity and prion binding properties

Salvia sclareoides is an aromatic herb native to Portugal, of which phenolic content (Folin–Ciocalteau method), chemical profile (HPLC/DAD), antioxidant activity (DPPH, β-carotene/linoleic acid assays), acute toxicity (MTT method, adapted for non-adherent cells), genotoxicity (short-term chromosomal aberration assay) and prion binding properties were evaluated in the acetone, water, ethanol, methanol and n-butanol extracts. The latter presented the highest phenolic content and antioxidant activity (DPPH assay), and was the single one with the flavonoids (+)-catechin, kaempferol O-glucoside and quercetin. Vanillic acid was the major component of all extracts but gallic, gentisic, caffeic, syringic, coumaric and ferulic acids were also found in some extracts. Only the n-butanol extract had components binding to the cellular form of human prion protein detected by NMR which showed specificity for two regions of the folded domain and for the unstructured N-terminal region. Extracts were not cytotoxic nor genotoxic, reinforcing the potential of S. sclareoides for nutraceutical purposes.     

Bone morphogenetic protein (BMP) ligands and receptors in bovine ovarian follicle cells: actions of BMP-4, -6 and -7 on granulosa cells and differential modulation of Smad-1 phosphorylation by follistatin

Given the paucity of information on the potential roles of bone morphogenetic proteins (BMPs) in the ruminant ovary we conducted immunolocalization and functional studies on cells isolated from bovine antral follicles. Immunocytochemistry revealed expression of BMP-4 and -7 in isolated theca cells whereas granulosa cells and oocytes selectively expressed BMP-6. All three cell types expressed a range of BMP-responsive type-I (BMPRIB, ActRI) and type-II (BMPRII, ActRII, ActRIIB) receptors supporting autocrine/paracrine roles within the follicle. This was reinforced by functional experiments on granulosa cells which showed that BMP-4, -6 and -7 promoted cellular accumulation of phosphorylated Smad-1 but not Smad-2 and enhanced 'basal' and IGF-stimulated secretion of oestradiol (E2), inhibin-A, activin-A and follistatin (FS). Concomitantly, each BMP suppressed 'basal' and IGF-stimulated progesterone secretion, consistent with an action to prevent or delay atresia and/or luteinization. BMPs also increased viable cell number under 'basal' (BMP-4 and -7) and IGF-stimulated (BMP-4, -6 and -7) conditions. Since FS, a product of bovine granulosa cells, has been shown to bind several BMPs, we used the Biacore technique to compare its binding affinities for activin-A (prototype FS ligand) and BMP-4, -6 and -7. Compared with activin-A (K(d) 0.28 +/- 0.02 nM; 100%), the relative affinities of FS for BMP-4, -6 and -7 were 10, 5 and 1% respectively. Moreover, studies on granulosa cells showed that preincubation of ligand with excess FS abolished activin-A-induced phosphorylation of Smad-2 and BMP-4-induced phosphorylation of Smad-1. However, FS only partially reversed BMP-6-induced Smad-1 phosphorylation and had no inhibitory effect on BMP-7-induced Smad-1 phosphorylation. These findings support functional roles for BMP-4, -6 and -7 as paracrine/autocrine modulators of granulosa cell steroidogenesis, peptide secretion and proliferation in bovine antral follicles. The finding that FS can differentially modulate BMP-induced receptor activation and that this correlates with the relative binding affinity of FS for each BMP type implicates FS as a potential modulator of BMP action in the ovary.