Towards the industrial production of antioxidants from food processing by-products with ultrasound-assisted extraction

Apple pomace, a by-product of the cider production, has been studied as a potential source of polyphenols, compounds of great interest for the industry. Ultrasound has been used to improve extraction efficiency in terms of time needed and total polyphenol content. A preliminary study has been first investigated to optimize ethanol proportion of aqueous extractant (50%, v/v) and solid/liquid ratio (<15%, w/v). A response surface methodology has then been used to maximize total polyphenol content of extracts and investigate influence of parameters involved in extraction procedures for both total polyphenols content and composition of extracts. Optimal settings reached from a central composite design were applied for ultrasound-assisted extraction and were compared to conventional procedure: yields were increased by more than 20%. Ultrasound-assisted polyphenols extraction from apple pomace appears to be a relevant, rapid, sustainable alternative to conventional procedure, and that scale up of the process is possible.     

A Method for In Vitro Measurement of Oxidized Low-Density Lipoprotein in Blood,Using Its Antibody,Fluorescence-Labeled Heptapeptide and Polyethylene Glycol

Two oxidized forms of low-density lipoprotein (LDL), oxidized (Ox-LDL) and minimally modified (MM-LDL), and the immune complexes (LDL-ICs) that they form with their corresponding antibodies, play a major role in the pathogenesis of atherosclerosis. Recently, we reported that the heptapeptide KP6 (Lys-Trp-Tyr-Lys-Asp-Gly-Asp) coupled through its ε-amino group present on the N-terminal Lys to fluorescein isothiocyanate (FITC)— (FITC)KP6— binds specifically to Ox-LDL and MM-LDL, but not to native LDL. Here, to develop a novel method for measuring the levels of oxidatively modified LDL in blood, using (FITC)KP6, we analyzed the latter’s binding with MM-LDL-IC and Ox-LDL-IC. Polyacrylamide gel electrophoresis analysis revealed that (FITC)KP6 could efficiently and specifically bind to polyethylene glycol (PEG)-precipitated MM-LDL-IC and Ox-LDL-IC in a dose-dependent manner with high sensitivity in plasma and serum. Our results indicate that the above method for measuring the levels of PEG-precipitated, oxidatively modified LDL-ICs, formed by the addition of anti-Ox-LDL antibody to blood, using (FITC)KP6, can aid the diagnosis of atherosclerosis.     

光谱法研究Cu~(2+)诱导的不同时间的LDL体外氧化

氧化低密度脂蛋白(oxidized low density lipoprotein,oxLDI)被认为是动脉硬化的关键致病因素.文章综合运用紫外吸收光谱、荧光光谱、同步荧光光谱及圆二色(circular diclaro‘lsm.CD)光谱研究了(好'诱导不同时间的U)IJ体外氧化过程.随着氧化时间的增加.生成的丙二醛(malonaldehyde,MDA)的量逐渐增加,其相对应的430 nm 处的荧光强度也随之加强,氧化过程中生成了新的氧化产物.氧化低密度脂蛋白紫外吸收增强;表征氨基酸残基信息的特征荧光强度和同步荧光强度降低;载脂蛋白B-100(apolipoproteinB-100,apoB-100)α-螺旋含量减少.实验结果表明,LDL氧化过程中Cu2+不仅诱导了氧化产物的生成,而且改变了apoB-100的构象进而导致了生色基团的微环境的变化.     

丁香有效部位对LDL中脂类、蛋白氧化修饰及光谱学变化的抑制

LDL氧化修饰是致动脉粥样硬化的关键性因素。其中,在氧化修饰过程中LDL所含脂类、蛋白及其光谱学性质均会受到影响。前期研究结果发现丁香对LDL氧化修饰具有很强的抑制作用。但是,丁香是否是通过抑制脂类及蛋白氧化来实现,以及丁香是否能抑制LDL氧化修饰后光谱学性质的改变,这些问题目前均不清楚。据此,运用紫外吸收光谱、荧光光谱等方法研究了丁香有效部位(effective fraction from clove,EFC)对LDL氧化修饰的抑制效果。研究结果表明,EFC能有效延缓脂质氧化过程中共轭二烯(CD)增殖及推后其达到最大值,并抑制胆固醇降解;也可抑制蛋白组分apoB100中Trp荧光猝灭及Lys氧化修饰和荧光光谱变化;同时也能抑制LDL中脂质与蛋白过氧化产物-脂褐素的生成;此外,该部位对LDL 氧化修饰过程中紫外-可见光谱学特征的改变也有影响。论文将为丁香抗AS功能食品研发提供参考。     

谷精草提取物中多酚含量的测定

以没食子酸为标准品,采用福林-酚(Folin-Ciocalteu)法测定多酚含量.结果表明,没食子酸在浓度为1-6μg/mL范围内与吸光度呈良好的线性关系(r=0.9999),谷精草提取物中多酚含量为5.58％,平均回收率为99.7％,相对标准偏差(RSD)为0.74％.该方法简便、灵敏、准确,可用于谷精草提取物中多酚含量的测定.     

Fluorescence and Ultraviolet Spectroscopic Evaluation of Phenolic Compounds,Antioxidant and Binding Activities in Some Kiwi Fruit Cultivars

Spectrophotometric assays (Folin–Ciocalteu, ferric-reducing/antioxidant power, cupric-reducing antioxidant capacity, and β-carotene linoleate model system) have been used to evaluate the total phenolics and flavonoids in kiwi fruit cultivars in ethanol and water extracts. Fluorescent and infrared measurements were correlated with the obtained spectroscopic data. It was found that the contents of the bioactive compounds and the level of antioxidant activity in different extracts differ significantly (p < 0.05). Bioactive compounds and antioxidant activities were significantly higher in two kiwi fruit cultivars (“Bidan” and “SKK12”) than in other studied samples. To our knowledge this is the first report showing the differences and similarities in new kiwi fruit cultivars, using these spectroscopic data. The ethanol extracts of these cultivars exhibited high binding properties with human serum albumin compared with rutin. In conclusion, the applied analytical methods showed the main compounds in the kiwi fruit cultivars and can be used for determination of these compounds in any plants. The relative knowledge would contribute to the pharmaceutical development and clinical application of extracts of kiwi fruit extracts.     

丁香对低密度脂蛋白氧化过程中荧光特性变化的影响

低密度脂蛋白(LDL)氧化修饰是导致动脉粥样硬化(AS)的关键因素,LDL在氧化修饰过程中荧光特性会发生一系列改变。为评价丁香对LDL氧化过程中荧光的抑制效果,采用传统荧光及三维荧光等高线光谱研究丁香对LDL氧化修饰过程中荧光特性的影响。结果显示： LDL氧化孵育荧光特性的最佳测定时间为48 h;丁香粗提物对LDL氧化修饰过程中色氨酸(Trp)荧光猝灭、荧光发色团红移、氧化产生的活性醛修饰apoB 100所含赖氨酸(Lys)残基变化、荧光物质和脂褐素产生及三维荧光的变化均具有较好的抑制作用,且抑制作用与粗提物浓度成正相关。在此基础上进一步发现,丁香各极性成分(正己烷相(丁香精油)、乙酸乙酯相、正丁醇相及水相)对LDL氧化修饰过程荧光特征的变化影响不一。其中,正己烷相对Trp荧光猝灭、荧光发色团红移及三维荧光变化的抑制作用最强,而乙酸乙酯相抑制活性醛修饰Lys残基、荧光物质和脂褐素的产生效果最好。结果表明,丁香对LDL氧化修饰过程中荧光特性的变化具有较好的抑制作用,抑制作用的主要物质集中在正己烷相和乙酸乙酯相。该研究为后续丁香组成分析及其功能食品研发提供参考。     

Folin-Ciocalteu比色法测定石榴皮提取物中的多酚

研究Folin-Ciocalteu比色法测定石榴皮提取物中多酚含量的优化条件,并对该方法的稳定性、精密度、重现性、回收率进行了实验。结果表明,石榴皮提取物中多酚含量测定的最佳条件为:Folin-Ciocalteu试剂1.00mL、10.0g/100mL Na2CO33.00mL、反应温度25℃、反应时间120min、测定波长672nm,所用没食子酸标准品在10—100μg/mL范围内与吸光度呈良好的线性关系(y=0.0098x+0.0148,R2=0.9982)。该法简便、快速、准确,是检测石榴皮提取物中总多酚的可靠方法。     

茶树叶与根表面的XPS表征

利用X射线光电子能谱(XPS)研究了茶树叶与根的表面化学组成与结构。结果表明,茶树表面主要由C, O, N和Al等四种元素组成,在茶树叶远轴面还有少量的P和F。通过查阅标准图谱数据库、参照木材表面XPS分析结果,对茶树表面测得的C1s结合能采用曲线拟合与分峰,得到三种形态：结合能为285 eV的是C1,来自C—C或C—H,代表角质、蜡质等脂类化合物;C2的结合能在286.35 eV（近轴面）和286.61 eV（远轴面）,是能够与氧形成单键的C—O,主要源于表面的纤维素;C3的结合能为288.04 eV（近轴面）和288.09 eV（远轴面）,为CO基团标志,综合N（1s）的结合能（399～401 eV）和O（1s）的分峰情况,为蛋白质的酰基标志。茶树根表面除含有与叶表面相同的C1(结合能285 eV)、C2（结合能286.49 eV）和C3（结合能288.78 eV）所代表的角质、蜡质、纤维素和蛋白质之外,还出现了结合能为283.32 eV的C5。由于其结合能低于C1,推测为茶树根表面的有机金属形态。茶树叶和根表面没有木材表面具有的羧基C4,说明根系分泌的有机酸游离存在于根表面。对茶树表面O（1s）图谱的拟合结果与C（1s）结果相吻合。计算不同C和O形态所占的比例得知,茶树叶面远轴面含氧基团多于近轴面,呈较高的氧化状态,因此远轴面化学性质较活泼;与叶相比,茶树根的角质和蜡质含量显著降低,含氧基团相对增多,因此化学性质较活泼,适于水分子和其他溶质分子通过。由于蛋白质含量为根表面＞远轴面＞近轴面,表明根表面的湿润度高于叶表面,而远轴面湿润度高于近轴面。存在于茶树表面Al的结合能均大于单质铝72.7 eV,在73.50 eV以上,为Al₂O₃形态,这将增大茶树表面的吸附作用。由于根表面Al₂O₃含量高于叶面,显示根具有更强的吸附能力。     

Selection of a cyclic nonapeptide inhibitor to α-chymotrypsin using a phage display peptide library

A cyclic nonapeptide library displayed on filamentous bacteriophages was selected 6 times against α-chymotrypsin (EC 3.4.21.1) at three different pH conditions (6.5, 7.0, and 7.5). Phage peptide clones from the sixth selection, at all three pH conditions, interacted more strongly with α-chymotrypsin than the original library and a wild-type phage did. DNA sequencing of the selected phage peptide clones showed that different cyclic nonapeptide sequences had been selected at the different pH conditions. The oxidized form of the synthetic peptide, Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys, selected at pH 7.5, could completely inhibit the enzymatic activity of α-chymotrypsin. The structurally related enzymes trypsin (bovine) and elastase (porcine) were only marginally inhibited by the same peptide under the same conditions. The inhibition constant for α-chymotrypsin was estimated to be 10^-6 M. Phage clones expressing this peptide had a lower affinity for phenylmethylsulfonylfluoride-modified α-chymotrypsin than for natural α-chymotrypsin as determined by an enzyme immunosorbent assay. This peptide phage clone was also competitively prevented from binding to α-chymotrypsin by the corresponding synthetic oxidized peptide. Collectively, the results suggest that the oxidized form of the selected peptide Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys interacts with the active site of α-chymotrypsin and acts as a specific inhibitor to the enzyme. To our knowledge, the selected sequence Cys-Cys-Phe-Ser-Trp-Arg-Cys-Arg-Cys has not been found in nature. This revised version was published online in August 2006 with corrections to the Cover Date.     

多酚与蛋白质相互作用的荧光内滤效应校正方法的选择

用荧光猝灭法研究猝灭剂与荧光供体的相互作用时,内滤效应会使分析结果不准确,需加以校正,其中物理与化学方法存在操作复杂、耗资大等缺点,而数学方法简便快捷,较易推广使用。采用荧光光谱法分析酚类物质与蛋白质的相互作用时内滤效应明显,然而目前针对酚类物质与蛋白质作用的荧光校正,国内外鲜见报道。以柿单宁级分(PT40)、A型连接ECG二聚体和EGCG为多酚化合物的代表,以中华眼镜蛇磷脂酶A₂(PLA₂)为蛋白模型,比较了四种数学公式的校正结果,以经典Stern-Volmer回归方程的K值、线性相关性及截距为评价标准,优选一种适用于多酚物质与蛋白质作用的荧光校正方法,并以PT40-PLA₂为例,分析校正前后的差异。结果表明,用Gauthier等建立的数学公式进行校正后,K值有所降低,线性相关性变好且截距更接近于1,此外用该式对PT40-PLA₂体系进行荧光校正后PT40与PLA₂相互作用的结合常数仅为校正前的60%,且对两者相互作用力类型的判断更加准确,推断该式更适合于多酚与蛋白质作用的荧光内滤效应校正。     

Visualization and Quantification of Vascular Structure of Fruit Using Magnetic Resonance Microimaging

Three-dimensional magnetic resonance (MR) images of four kinds of fruit (Japanese apricot, peach, Japanese pear, and apple) were acquired throughout their growing periods (from April to August) to visualize and quantify their vascular structures. The vascular structures were emphasized using T ₁-weighted spin-echo or gradient-echo pulse sequences, visualized using a maximum-intensity projection technique, and quantified using an image-segmentation program. As a result, we demonstrated that the vascular volume changed almost in proportion to the whole volume and that larger fruits generally had larger proportionality constants. We therefore concluded that MR microimaging is a powerful tool for studies of the vascular structure of fruit.     

Do ultrasonic field effects upon the polyphenolics profile of propolis extracts improve their antioxidant and antimicrobial activity?

Ultrasound-assisted extraction (UAE) was applied for polyphenols extraction from Romanian propolis, followed by comparison with previous maceration work. The effects consisted not only in time reduction and extraction yield increase, but also in polyphenolics profile modification in terms of flavonoids / polyphenolic acids ratio. The operating parameters were ultrasounds (US) field exposure time (10–100 min), solvent composition (water, 25 % and 50 % ethanolic solutions, w/w), and liquid:solid ratio (2:1, 4:1 and 6:1, w:w), while keeping temperature constant. 24 polyphenolic derivatives were quantified by UHPLC-HRMS. UAE favored the extraction of pinocembrin, isorhamnetin and chrysin in water and 25 % ethanol, leading to different profiles than maceration, and further influences upon the antioxidant and antimicrobial activity. All extracts demonstrated increased antibacterial and antifungal activity compared to maceration, particularly the 50 % ethanolic extracts, which presented a three-times larger antioxidant capacity. Chemometric methods (Principal Component Analysis – PCA and Partial Least Squares Regression – PLS) and a saturation type model were used to correlate the polyphenolics profiles and antioxidant capacity. Experimental and modelling results concluded that 50 % ethanolic solutions and UAE represent the favorable operating conditions in terms of yield and extracts quality.     

Ultrasound-alkaline combined extraction improves the release of bound polyphenols from pitahaya (Hylocereus undatus 'Foo-Lon') peel: Composition,antioxidant activities and enzyme inhibitory activity

In this study, ultrasound-assisted alkaline hydrolysis was used to extract polyphenols from pitahaya peel. The effects of sonication time, ultrasonic density, NaOH concentration and the liquid–material ratio on the total phenolic content (TPC), total flavonoid content (TFC) and antioxidant activity of the extracts were studied. The composition and content difference of the extracts were analyzed and the inhibitory effect of α-amylase and α-glucosidase was measured. The results of single-factor analysis showed that when the sonication time was 45 min, the ultrasonic density was 32 W/L, the NaOH solution concentration was 6 M and the liquid–material ratio was 30 mL/g, the release of phenolic compounds was the largest and the antioxidant activity was the strongest. An UPLC-QTOF-MS/MS method was used to analyze the components and contents of the extracts. We found that there was a great difference in the component content of the free polyphenol extract and the bound polyphenol extract. From the results, we concluded that there was a strong correlation between the type and content of phenolic compounds and antioxidant activities, indicating that phenolic compounds were the main compounds of these biological activities. Moreover, the bound polyphenol extracts showed a significant inhibitory effect on α-amylase and α-glucosidase was stronger than that of the free polyphenol extracts. In addition, scanning electron microscopy showed that ultrasound-assisted extraction is crucial to the destruction of the cell wall and the release of bound polyphenols. Therefore, the pitahaya peel has the potential for therapeutic, nutritional, and functional food applications, and ultrasound-assisted alkaline hydrolysis is an effective means to release phenolic compounds.     

Atomic structure sensitivity of XPS: The oxidation of cobalt

The cobalt (0001) surface has been oxidized to form an epitaxial CoO structure. The intraplaner lattice parameter of the CoO surface has been measured by LEED to be initially 8% larger than that of the bulk and to approach that of the bulk with increasing oxygen exposure. This lattice parameter change has been related to an observed 0.5 eV shift to higher binding energy shoulder on the oxygen 1s level as a function of oxygen exposure. A high binding energy shoulder on the oxygen 1s level has also been identified as non-stoichiometric oxygen in the cobalt oxide.     

基于光谱学技术对丁香油与乙酸乙酯相抑LDL非酶糖基化、氧化修饰能力的比较

低密度脂蛋白(LDL)非酶糖基化和氧化修饰均是导致动脉粥样硬化(AS)的关键因素。前人发现,丁香可有效抑制牛血清白蛋白(BSA)、高密度脂蛋白(HDL)糖基化修饰,丁香乙酸乙酯相(EAEC)和丁香油(CBO)均具有很强的抗氧化活性。为了确定丁香抑制LDL糖基化及氧化所对应的最有效成分,在前人研究基础上采用光谱学技术对EAEC和CBO抑制LDL糖基化及氧化功能进行了比较。首先建立LDL-葡萄糖糖基化孵育体系,通过测定糖基化早期、中期和末期产物及三维荧光光谱的变化比较CBO和EAEC抑制LDL糖基化修饰的效果。其次,建立LDL-CuSO₄氧化孵育体系,通过测定荧光指标和三维荧光光谱、紫外可见波长扫描来比较两者抑制LDL氧化修饰效果。结果发现,EAEC和CBO对LDL糖基化修饰过程中产生的早期产物(Amadori产物)、中期产物(二羰基化合物)及终末期产物(戊糖素和AGEs)均具有很强的抑制效果,且EAEC的抑制效果均强于CBO;三维荧光光谱表征得到相同的结果。在LDL氧化修饰比较中,CBO对色氨酸(Trp)荧光猝灭、总荧光产物及脂褐素的生成、赖氨酸(Lys)修饰的抑制效果均显著强于非油成分的EAEC;三维荧光光谱表征也得到相同的结果;紫外、可见全波长扫描反映CBO比EAEC对共轭二烯(CD)生成及光谱红移的抑制效果更强。研究结果为后续锁定重点成分分离、纯化及研发丁香抗LDL糖基化及氧化功能食品提供参考。     

荧光光谱法和分子对接模拟技术研究白藜芦醇与胃蛋白酶的相互作用

白藜芦醇（Resveratrol, RES）属于非黄酮类多酚化合物,存在于葡萄科、百合科等多种植物体内,是一种具有多种生物活性和药理作用的天然活性物质,被广泛应用于食品和药品领域。研究表明多酚在生物体消化吸收过程中,会与消化酶（如胃蛋白酶、胰蛋白酶等）相互作用,使多酚类物质和消化酶的生物活性发生改变,进而影响多酚物质和其他营养物质的消化吸收,而RES与胃蛋白酶(Pepsin, PEP)的分子间相互作用机制未见报道。采用荧光光谱、紫外-可见吸收光谱、红外光谱和分子对接模拟等技术研究不同温度下RES与PEP相互作用的结合特性,为阐明RES和PEP的作用机制提供重要信息,同时为RES在食品和药品领域的应用提供理论参考。荧光光谱实验结果表明,PEP的荧光强度随着RES浓度的增加呈现出有规律的降低,表明RES对PEP有荧光猝灭作用。加入RES前后,PEP的紫外吸收光谱发生明显变化,初步判断RES与PEP的相互作用属于静态荧光猝灭类型;根据Stern-Volmer方程计算得到不同温度下最小猝灭速率常数K_q值远大于猝灭剂对生物大分子的最大扩散碰撞常数2.0×1010 L·mol-1·s-1,且猝灭常数(K_SV)与温度呈负相关关系,进一步验证了RES与PEP静态荧光猝灭类型结论。化学计量结合的值数目大约等于1,表明一个RES分子只能结合一个PEP分子。根据Van’t Hoff 方程以及热力学方程计算得到结果显示,ΔG＜0,说明RES与PEP的结合过程可以自发进行;ΔH＜0和ΔS＜0,表明RES与PEP之间结合作用力类型主要是氢键和范德华力。RES与PEP相互作用的同步荧光光谱和三维荧光光谱图表明,在RES的作用下,PEP的构象和微环境发生变化,色氨酸或酪氨酸残基所处微环境极性增强,疏水性减弱,蛋白构象变得疏松。红外光谱显示RES能使PEP的二级结构中α螺旋含量降低,β折叠含量增加,β转角和无规则卷曲变化不明显,这可能会影响PEP的活性。分子对接模拟实验结果显示RES与PEP中的残基Asp-32,Gly-34,Ser-35,Asn-37,Tyr-75,Gly-76,Thr-77,Ile-128,Ala-130及Gly-217有范德华力作用,与残基Ile-128及Asp-215产生超共轭效应,与残基Ser-36,Asn-37,Ile-128及Thr-218形成氢键,各种作用力使RES与PEP形成较稳定的复合物。     

Structural effect on degradability and in vivo contrast enhancement of polydisulfide Gd(III) complexes as biodegradable macromolecular MRI contrast agents

The structural effect of biodegradable macromolecular magnetic resonance imaging (MRI) contrast agents, polydisulfide gadolinium (Gd)(III) chelates, on their in vitro degradability, and cardiovascular and tumor imaging were evaluated in mice. Polydisulfide Gd(III) chelates, Gd-DTPA cystamine copolymers (GDCC), Gd-DTPA l-cystine copolymers (GDCP), Gd-DTPA d-cystine copolymers (dGDCP) and Gd-DTPA glutathione (oxidized) copolymers (GDGP), with different sizes and narrow molecular weight distribution were prepared and evaluated both in vitro and in vivo in mice bearing MDA-MB-231 tumor xenografts. GDGP with large steric hindrance around the disulfide bonds had greater T(1) and T(2) relaxivities than GDCC, GDCP and dGDCP. The degradability of the polydisulfide by the endogenous thiols decreased with increasing steric effects around the disulfide bonds in the order of GDCC>GDCP, dGDCP>GDGP. The size and degradability of the contrast agents had a significant impact on vascular contrast enhancement kinetics. The agents with a large size and low degradability resulted in more prolonged vascular enhancement than the agents with a small size and high degradability. It seems that the size and degradability of the agents did not significantly affect tumor enhancement. All agents resulted in significant contrast enhancement in tumor tissue. This study has demonstrated that the vascular enhancement kinetics of the polydisulfide MRI contrast agents can be controlled by their sizes and structures. The polydisulfide Gd(III) chelates are promising biodegradable macromolecular MRI contrast agents for magnetic resonance angiography and cancer imaging.     

Nogo-B诱导oxLDL降解与肝癌基因激活

本文基于Hill动力学与Michaelis-Menten方程,建立理论模型研究内质网定位的蛋白Nogo-B诱导调节氧化修饰低密度脂蛋白(oxidized low density lipoprotein(oxLDL))降解与肝癌基因激活.理论模型考虑oxLDL*(降解的oxLDL)-Nogo-B-Yes-associated protein (YAP)通路,研究发现,oxLDL的降解,促进了大量的Lysopho-sphatidic acid (LPA)产生,之后便会提高Hippo信号通路YAP活性,激活了癌基因的表达;经过约5小时Nogo-B表达上调,Nogo-B决定着Nogo-B与Autophagy-related 5 gene(ATG5)的复合体NA,NA调控oxLDL的降解,未降解的oxLDL会诱导Nogo-B表达上调,激活了oxLDL*-Nogo-B-YAP通路,理论结果符合实验结果,并揭示非酒精性脂肪肝病诱发的肝癌的致病机理,可以为设计阻断肝炎向肝癌转变的通路治疗方案提供理论依据.