基于毛细管电泳的黄芪抗氧化生物指纹图谱研究

根据评价抗氧化剂清除自由基能力的间接化学发光法,结合具有高效分离能力的毛细管电泳技术,建立了能表征中药抗氧化活性的生物指纹图谱。选取中药黄芪为研究对象,以黄芪甲苷为参照物峰,确定了6个共有峰,并首次测得黄芪甲苷对羟自由基的清除率。各共有峰相对迁移时间的RSD<1.5%,相对自由基清除率的RSD<8.4%,表明方法的精密度和重复性较好。7个产地的黄芪药材与道地药材具有良好的抗氧化活性相似度,相似度>96.5%。抗氧化生物指纹图谱对天然产物的质量控制与临床研究具有指导意义。     

褪色光度法测定Fenton反应产生的羟自由基及其应用

建立检测Fenton反应产生羟自由基的新方法。Fenton反应产生的羟自由基与苋菜红反应，颜色发生变化，用分光光度计测定其△A510值的变化，可间接测定羟自由基的生成量。通过对测定条件的研究，确定了体系最佳实验条件。抗氧化药物甘露醇、硫脲与羟自由基清除率具有明显的量效关系。测定了阿魏酸、芦丁等几种中药活性成分清除羟自由基的功能，此法可用于羟自由基清除剂的筛选及抗羟自由基机理研究。     

罗丹明6G-锰(Ⅱ)-过氧化体系荧光法测定常见油性种子的抗氧化活性

碱性介质中罗丹明6G能产生特征荧光,其最大激发波长和发射波长分别为350 nm和550 nm;Mn2+-H2O2体系在碱性介质中产生的羟自由基可以迅速氧化罗丹明6G使其荧光猝灭,而油性种子的浸提物可以清除羟自由基,从而使溶液的荧光猝灭程度降低,据此建立了测定油性种子抗氧化性的新方法.实验观察到抗氧化剂维生素C和硫脲等在低浓度范围内对羟自由基的清除率与用量呈上升关系,而在大浓度下反而下降.讨论了样品的水、醋酸、乙醇溶剂浸提物对羟自由基的清除效率.用水、醋酸作提取剂,分别测试了7种常见油性种子的抗氧化活性.     

苯甲酸-Fenton体系荧光法测定中草药抗氧化活性

利用Feton体系产生羟自由基,能与苯甲酸反应生成具有强荧光的产物,加入抗氧化剂可有效清除羟自由基,导致体系荧光强度降低.据此建立了筛选抗氧化剂的荧光新体系.应用所建立的方法测定了葛根、吴茱萸和黄连三种常见中草药的抗氧化活性.结果表明,当中草药的浓度为0.4 mg(dry weight)/mL时,葛根、吴茱萸、黄连对羟...     

罗丹明6G-锰（Ⅱ）-过氧化氢体系荧光法测定常见油性种子的抗氧化活性

碱性介质中罗丹明6G能产生特征荧光，其最大激发波长和发射波长分别为350nm和550nm；Mn^2 -H2O2体系在碱性介质中产生的羟自由基可以迅速氧化罗丹明6G使其荧光猝灭，而油性种子的浸提物可以清除羟自由基，从而使溶液的荧光猝灭程度降低，据此建立了测定油性种子抗氧化性的新方法。实验观察到：抗氧化剂维生素C和硫脲等在低浓度范围内对羟自由基的清除率与用量呈上升关系，而在大浓度下反而下降。讨论了样品的水、醋酸、乙醇溶剂浸提物对羟自由基的清除效率。用水、醋酸作提取剂，分别测试了7种常见油性种子的抗氧化活性。     

矮垂头菊不同极性溶剂萃取物的抗氧化活性研究

为了研究矮垂头菊植物的抗氧化活性,本文采用DPPH自由基清除法、ABTS自由基清除法、羟基自由基清除法、超氧自由基清除法、一氧化氮自由基清除法和铁还原/抗氧化能力(FRAP)测定法六种抗氧化对矮垂头菊粗提物(CE)、正己烷相(HE)、乙酸乙酯相(EAE)、正丁醇相(BE)、水相(WE)等5个不同极性溶剂萃取物的抗氧化活性进行评价,并探讨抗氧化活性与总酚和总黄酮含量之间的关系。结果显示矮垂头菊不同极性溶剂的萃取物均有抗氧化活性,且抗氧化活性与总黄酮和总酚含量密切相关,其中BE中总黄酮含量最高可达588.02±10.18 mg芦丁/g,总酚含量最高可达568.49±8.14 mg没食子酸/g,且自由基清除能力和FRAP值均高于其它组分,但HE中总黄酮和总酚含量及抗氧化活性均较低。结果表明矮垂头菊EAE萃取物和BE萃取物具有很强的自由基清除能力,可作为潜在的天然抗氧化剂开发。     

Ag(Ⅲ)-鲁米诺流动注射化学发光法用于茶饮料抗氧化活性的评价

以茶叶中常见的8种茶多酚化合物为研究对象,研究了三价银配合物[Ag(Ⅲ)]-鲁米诺流动注射化学发光法与2,2-联苯基-1-苦基肼基(DPPH)自由基清除法、邻苯三酚-碳酸盐-鲁米诺化学发光法和鲁米诺-过氧化氢化学发光法检测结果的相关性,并采用Ag(Ⅲ)-鲁米诺流动注射化学发光法评价了4种茶饮料的抗氧化活性。设置的仪器工作参数如下:载流为纯水,蠕动泵转速为25r·min~(-1),光电倍增管负高压为600V。结果发现:Ag(Ⅲ)-鲁米诺流动注射化学发光法所得化学发光强度差值与DPPH自由基清除法所得的IC_(50)(自由基清除率为50%时对应的抗氧化剂浓度)相关性较好,相关系数为0.949 0。方法用于市售4种茶饮料抗氧化性能评价,所得结果与DPPH自由基清除法一致,相关系数为0.966 5。     

芜菁籽不同溶剂提取物体外抗氧化活性研究

通过体外试验初步研究芜菁籽提取物的抗氧化活性。用95%乙醇为溶剂提取芜菁籽,采用有机溶剂萃取法将提取物分为石油醚相,乙酸乙酯相,正丁醇相和水相等4个不同极性部位、同时测定了各相提取物对羟自由基,超氧阴离子自由基,DPPH自由基的清除能力,并与合成抗氧化剂BHT进行对照。结果表明,芜菁籽提取物的不同极性部位均有抗氧化活性,其抗氧化效果随着浓度的增加而增强,乙酸乙酯相和正丁醇相的还原能力及清除O-2.,DPPH和.OH的能力均强于水相和石油醚相,乙酸乙酯相和正丁醇相的抗氧化作用强,是天然抗氧化剂的良好来源,应对其进一步分离纯化。     

基于果糖自氧化体系的抗氧化剂筛选方法

机体内果糖的自氧化过程中会产生多种自由基, 并最终转化为羟自由基, 苯甲酸钠可捕获羟自由基生成具有强荧光信号的羟基苯甲酸钠. 本文采用荧光光度法考察了影响果糖自氧化体系的各种因素, 建立了果糖自氧化产生羟自由基体系. 实验结果表明, 在果糖浓度为8.00 mmol/L, CuSO₄浓度为20.0 μmol/L, 苯甲酸钠浓度为24.0 mmol/L, pH=7.4, 温度为37℃及反应时间为24 h的条件下, 果糖自氧化体系最终可产生19.27 μmol/L的羟自由基. 抗氧化剂的存在可清除果糖自氧化过程中产生的自由基, 使最终生成的羟自由基的量减少, 从而导致生成的羟基苯甲酸钠减少, 荧光信号减弱, 由此建立了基于果糖自氧化体系的抗氧化剂筛选方法. 利用本评价体系考察了抗氧化剂盐酸小檗碱和阿魏酸的抗氧化能力, 实验结果表明, 中药标准品盐酸小檗碱和阿魏酸均能有效清除果糖自氧化体系产生的羟自由基, 其IC₅₀值分别为0.023和0.036 mmol/L.     

4种黄酮小分子对DPPH自由基的清除作用及构效关系研究

通过紫外可见光谱测定了4种黄酮小分子芦丁、牡荆素、山奈素、金丝桃苷对DPPH自由基的清除率、稳定性及半抑制浓度(IC50),并以常用的天然抗氧化剂抗坏血酸作为对照,考察了其抗氧化效果,探讨了黄酮类化合物的抗氧化性与结构的关系。结果表明：不同的抗氧化剂清除DPPH自由基达到平衡的时间不同,芦丁所需时间最长。4种黄酮小分子及抗坏血酸均对DPPH自由基有清除效果,并存在一定的量效关系。对DPPH自由基的清除能力从大到小依次为金丝桃苷、抗坏血酸、芦丁、山奈素、牡荆素。结构分析表明,B环邻二酚羟基是黄酮类化合物抗氧化所必需的基团,其羟甲基化及A环羟基糖苷化不利于黄酮类化合物的抗氧化活性。而C环3-OH的糖苷化对抗氧化活性有利,且单糖苷优于双糖苷。     

Isolation and Purification of Highly Polar Antioxidants from Chirita longgangensis by Combination of Macroporous Resin and HSCCC

An efficient and simple method to isolate and purify highly polar antioxidants from the antioxidant active site of Chirita longgangensishas been established. Firstly, the antioxidant active site was enriched with D101 macroporous resin, and then high-speed counter-current chromatography (HSCCC) was used with the two-phase solvent system ethyl acetate–n-butanol–methanol–water (5:0.1:0.5:4.5, v/v) to obtain four antioxidants in one step. They were identified as plantainoside D (28.4 mg), plantainoside B (9.5 mg), calcedarioside B (18.1 mg) and calcedarioside A (16.7 mg) by analysis of electrospray ionization mass spectrometry (ESI-MS) and nuclear magnetic resonance (NMR) spectra. The purities were all above 97 % as determined by HPLC. The inhibiting effects of the crude extracts, enriched fraction and the obtained compounds on superoxide anion radical, hydroxyl radical and hydrogen peroxide were determined by different chemiluminescence (CL) systems. The result shows that all of them have good antioxidant activity. However, the sequence of antioxidant abilities among compounds I–IV was different when assayed by different CL systems (superoxide anion radical, hydroxyl radical, and hydrogen peroxide). This is the first report on preparative isolation and purification of antioxidants from C. longgangensis by HSCCC combined with macroporous resin and their inhibition of free radical-induced luminol chemiluminescence.     

Antioxidant activity of aryltetralone lignans and derivatives from Virola sebifera (Aubl.)

Aryltetralone lignans bearing methylenedioxy groups (1a-b; 2a-b) were isolated from seeds of Virola sebifera. Their antioxidant activities were evaluated by inhibition of lipid peroxidation as indicated by TBARS and chemiluminescence emission (CL) assays. The lignan 1c, 'having a 2'-hydroxy-4',5'-methylenedioxyphenyl group, was the most active compound with TBARS/CL Q 1/2 values of 0.89 and 0.10 microg/mL, respectively. The catechol derivatives 3 and 4, obtained by demethylenation of lignans 1a and 2a, were of similar activity to 1c, and all were much more effective as antioxidants than alpha-tocopherol.     

On-line screening methods for antioxidants scavenging superoxide anion radical and hydrogen peroxide by liquid chromatography with indirect chemiluminescence detection

The identification of radical species is possible by the electron spin resonance technique. However, the antioxidants in complex matrices such as biological and food samples are difficult to determine. Hence, we developed novel screening systems for antioxidants, which are mainly eliminating superoxide anion radical (O(2)(-)) and hydrogen peroxide (H(2)O(2)), by HPLC with luminol-based chemiluminescence (CL) detection. When the sample contains antioxidants, inhibited peaks corresponding to each antioxidant are observed on the chromatogram. The antioxidant activities of catechins and flavones were determined with flow injection analysis by the proposed indirect CL. The scavenging activity for H(2)O(2) and O(2)(-) were different from each catechin and flavone. Furthermore, the potential was dependent upon the number and the position of OH functional group in the structure. Some applications such as the screening of antioxidants in tea products were also investigated. In spite of many peaks appeared on the chromatogram at UV detection, only the peaks corresponding to the compounds having elimination effect to O(2)(-) and/or H(2)O(2) were detected as inhibited peaks. Consequently, the proposed HPLC-CL seems to provide new screening systems for antioxidants possessing inhibition activity of O(2)(-) and H(2)O(2).     

Optical Fiber Chemiluminescence Biosensor for Antioxidants Based on an Immobilized Luminol/Hematin Reagent Phase

ABSTRACT

A chemiluminescence (CL) biosensing system for antioxidants was developed based on luminol and hematin co-immobilized on a cellulose membrane disc. The concentration of the antioxidant was quantified through the measurement of the inhibition of the CL emitted when hydrogen peroxide was introduced into the reagent phase. The instrumentation employed in the measurement was a fabricated luminometer employing optical fibers and a UV-enhanced photodiode transducer. Under optimum conditions, linear calibration curves were obtained for antioxidant concentrations ranging from 1 × 10^-4 M to 0.10 M, with an average relative standard deviation of about 5%. The minimum detectable concentration was 100 μM, and the response time was less than 60 seconds. The sensor response correlated well (r = 0.9979) with the results of a standard colorimetric method for a specific antioxidant (propyl gallate). The sensor was used to assess the antioxidant capacity of water infusions prepared from the dried leaves of some Philippine medicinal plants.     

Chemiluminescence from poly(styrene-b-ethylene-co-butylene-b-styrene) (SEBS) block copolymers

A detailed analysis of the chemiluminescence emission (CL) from poly(styrene-b-ethylene-co-butylene-b-styrene), SEBS, was carried out. A phenol-phosphite stabilization system based on Irgafos 168 and Irganox 1330, was studied. The kinetic analysis of the CL profile under nitrogen shows a first-order reaction for the decay of chemiluminescence. The activation energy shows different values as a function of temperature, showing that different reactions are involved in the thermal degradation of the SEBS. The CL decay rate correlates well with the amount of the phosphite, Irgafos 168, and confirms the activity of this stabilizer as radical chain-breaking antioxidant in these copolymers.The isothermal analysis of CL under oxygen allows evaluation of the oxidation state, as well as the efficiency of the antioxidants. Good correlations are found between the CL parameters and concentration of Irgafos 168. Several factors suggest that oxidation begins in the interfacial region. Spectral analysis of the chemiluminescence shows the presence of different types of hydroperoxides.Finally, the characterization of the SEBS copolymers by differential scanning calorimetry reveals an order-disorder transition, assigned to aggregates that behave as paracrystalline regions.     

Sequential injection analysis with chemiluminescence detection for the antioxidative activity against singlet oxygen.

A sequential injection analysis (SIA) with chemiluminescence (CL) detection was developed for the measurement of antioxidative activity against singlet oxygen ((1)O2). Lactoperoxidase-hydrogen peroxide-bromide ion system was used for the generation of (1)O2. When a 100 mM sodium acetate buffer (pH 4.5) was used as a carrier solution, the SIA-CL system could be optimized with respect to the flow-rate of the carrier, concentration of reagents and their aspiration order. The antioxidative activity was expressed as an attenuation of luminol CL due to the quenching of (1)O2 by an antioxidant. The relative standard deviations of antioxidative activity (n=3) against (1)O2 for within- and between-day analyses were < or = 1.6% (20 microM Trolox). The system was successfully applied to the assay of antioxidative activities of various antioxidants including vitamin supplements at a rate of 10 samples within 15 min. The proposed SIA-CL system was rapid and reproducible with minimum consumption of the sample and of reagents, and thus was useful for the screening of compounds possessing antioxidative activity against (1)O2.     

Development and validation of a sequential-injection method with chemiluminescence detection for the high throughput assay of the total antioxidant capacity of wines

This work reports a sequential-injection analysis (SIA) method with chemiluminescence (CL) detection for the rapid assay of the total antioxidant capacity (TAC) in wines. The method exploited the Co(II)-catalysed CL reaction of luminol with hydrogen peroxide in alkaline medium. Zones of sample, hydrogen peroxide, catalyst (Co(II) solution) and alkaline luminol were sequentially aspirated into the holding coil of the SIA manifold. Then, the flow was reversed and the stacked zones were directed to the CL detector. As the zones overlapped, antioxidants in the samples scavenged a portion of hydrogen peroxide and the decrease in the CL intensity was monitored and related to the TAC. The chemical and geometric conditions were studied and the method was validated in terms of linearity, accuracy (trueness and precision), matrix effects, signal additivity and robustness. The reproducibility of the method (expressed as the between-days % relative standard deviation) was between 2.5 and 3.4% and the trueness (expressed as the % recovery in wines spiked with gallic acid) was in the range 96.7-97.3%. The sampling frequency was 60 samples h⁻¹. The proposed SIA-CL method was compared with the DPPH method and the Folin-Ciocalteau (FC) method for the analysis of 25 wine samples.     

Antioxidant effects of dihydroquercetin and rutin in peroxidase reactions catalyzed by cytochrome <Emphasis Type="Italic">c</Emphasis>

The combination of cytochrome c (Cyt c) and cardiolipin is known to catalyze free radical oxidation of membrane lipids, thereby triggering the cascade of reactions that lead to programmed cell death (apoptosis). It was interesting to estimate the possibility of controlling this reaction with the use of inhibitors of free radical reactions (antioxidants). The effects of bovine heart cardiolipin (BCL) and synthetic 1,1′,2,2′-tetraoleyl cardiolipin (TOCL) on the Cyt c peroxidase activity detected by luminol-enhanced chemiluminescence (CL) were studied. The CL amplitude substantially increased in the presence of TOCL and BCL in the ranges of the cardiolipin to Cyt c molar ratios from 0 to 30 and from 0 to 50, respectively, and insignificantly increased upon further increase in these ratios to 640: 1 and 320: 1. This dependence of the CL amplitude on the cardiolipin to protein ratio may have been accounted for by saturation of the protein surface with cardiolipin molecules reached at a certain molar ratio. The effects of the antioxidants rutin and dihydroquercetin (DHQ) on the peroxidase activity of Cyt c in combination with BCL was studied in the presence of the CL activator coumarine C-525 at a BCL to Cyt c molar ratio of 32: 1. In both cases, the peroxidase activity decreased as the antioxidant content of the system increased. A 50% inhibition of the reaction was observed at DHQ and rutin concentrations of 10 and 3 μM, respectively. It is supposed that the use of antioxidants will make it possible to control the formation of lipid radicals on membrane-bound Cyt c, which is the key stage of apoptosis.     

Chemiluminescence of polyethylene: The comparative antioxidant effectiveness of phenolic stabilizers in low‐density polyethylene

The thermal stabilizing efficiency of a range of phenolic antioxidants (Lowinox CA22, Lowinox WSP, Lowinox TBP6, Irganox 3114, Irganox 1330, and Cyanox 1790) was determined in polyethylene films with chemiluminescence and hydroperoxide analysis and compared with standard systems based on Irganox 1010 and 1076. Under both nitrogen and oxygen conditions, good correlations were obtained between the two methods, confirming the importance and role of the hydroperoxide functionality and its stability in the oxidative process. Chemiluminescence decay rates correlated well with the initial corresponding hydroperoxide contents, which were measures of the antioxidant efficiencies in the polymer. The antioxidant structure and volatility (melting points) were important parameters to consider in any such correlations and related very much to the methodology and conditions of analysis (i.e., the temperature and atmosphere). Some of the antioxidants themselves under nitrogen exhibited strong chemiluminescence, which appeared to be a legacy associated with their manufacturing history and the partial oxidation of their structures, which gave peroxide functionalities. This was more notable for the complex antioxidant structures. Under oxygen, higher levels of chemiluminescence were observed, and this was indicative of some level of oxidation associated with the antioxidant structures. With temperature‐ramping experiments, the chemiluminescence emission was significant and only observed at temperatures close to the melting points of the additives and/or polymer. Mobility was clearly an essential feature of this reaction emission because chemiluminescence was well observed when the molten state was reached. Under normal practical conditions, such levels of chemiluminescence, because of employed stabilizers, do not contribute significantly to the chemiluminescence emissions of stabilized polymer materials. © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 3312–3326, 2002