Interaction of pyrogallol red with peroxyl radicals. A basis for a simple methodology for the evaluation of antioxidant capabilities

A competitive method to evaluate the reactivity of highly reactive antioxidants is reported. Pyrogallol red (PGR) and AAPH (2,2'-azo-bis-(2-amidinopropane) dihydrochloride) were employed as target-molecule and peroxyl radical source, respectively. In the zero-order kinetic limit in PGR, the dependence of the ratio R(o)/R (where R(o) is the rate of the process in the absence of additive and R is the rate of the process in the presence of additive) upon the additive concentration (Stern-Volmer like plots) was studied. Various polyphenols (n=10) and ascorbic acid (AA) were tested as additives. In PGR protection by AA, was observed a neat induction time, associated to the total protection of the target molecule. On the other hand, the experiments that were carried out in presence of phenolic compounds allowed a relative evaluation of their reactivity towards peroxyl radicals. This reactivity follows the order quercetin > gallic acid > Trolox > kaempferol. Data obtained employing quercetin and Trolox are compatible with a competitive protection by these antioxidants. Due to the high reactivity of PGR towards peroxyl radicals and its high extinction coefficient at long wavelengths, it is a very suitable molecule to be employed as target in the evaluation of the free radical scavenging capability of very reactive phenolic compounds.     

Oxidative modification of citrate synthase by peroxyl radicals and protection with novel antioxidants

In mammals, aging is linked to a decline in the activity of citrate synthase (CS; E.C. 2.3.3.1), the first enzyme of the citric acid cycle. We used 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), a water-soluble generator of peroxyl and alkoxyl radicals, to investigate the susceptibility of CS to oxidative damage. Treatment of isolated mitochondria with AAPH for 8–24?h led to CS inactivation; however, the activity of aconitase, a mitochondrial enzyme routinely used as an oxidative stress marker, was unaffected. In addition to enzyme inactivation, AAPH treatment of purified CS resulted in dityrosine formation, increased protein surface hydrophobicity, and loss of tryptophan fluorescence. Propyl gallate, 1,8-naphthalenediol, 2,3-naphthalenediol, ascorbic acid, glutathione, and oxaloacetate protected CS from AAPH-mediated inactivation, with IC₅₀ values of 9, 14, 34, 37, 150, and 160?μM, respectively. Surprisingly, the antioxidant epigallocatechin gallate offered no protection against AAPH, but instead caused CS inactivation. Our results suggest that the current practice of using the enzymatic activity of CS as an index of mitochondrial abundance and the use of aconitase activity as an oxidative stress marker may be inappropriate, especially in oxidative stress-related studies, during which alkyl peroxyl and alkoxyl radicals can be generated.     

Pyrogallol inhibits the growth of human pulmonary adenocarcinoma A549 cells by arresting cell cycle and triggering apoptosis

Pyrogallol (PG) is a polyphenol compound and has been known to be an O generator. We evaluated the effects of PG on the growth of human pulmonary A549 cells in relation to the cell cycle and apoptosis. Treatment with 50 or 100 μM PG significantly inhibited the cell growth of A549 for 72 h. DNA flow cytometric analysis indicated that PG slightly induced a G1 phase arrest of the cell cycle at 24 or 48 h, but did not induce the specific cell cycle arrest at 72 h. Intracellular GSH depletion was observed in PG‐treated cells. PG induced apoptosis in A549 cells, as evidenced by sub‐G1 cells, annexin V staining cells, and the loss of mitochondrial membrane potential (Δ Ψ_m). The intracellular ROS (reactive oxygen species) level including O increased in PG‐treated A549 cells at 24 and 48 h, and persisted at 72 h. The changes in GSH as well as ROS levels by PG affected the cell viability in A549 cells. In conclusion, PG inhibited the growth of human pulmonary A549 cells by inducing cell cycle arrest as well as triggering apoptosis. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:36–42, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20263     

Peroxyl radical is produced upon the interaction of hypochlorite with tert-butyl hydroperoxide

As we reported previously, hypochlorite interacting with organic hydroperoxides causes their decomposition ((1995) Biochemistry (Moscow), 60, 1079-1086). This interaction was supposed to be a free-radical process and serve as a source of free radicals initiating lipid peroxidation (LP). The present study is the first attempt to detect and identify free radicals produced in the reaction of hypochlorite with tert-butyl hydroperoxide, (CH₃)₃COOH, which we have used as an example of organic hydroperoxides. We have used a direct method for free radical detection, EPR of spin trapping, and the following spin traps: N-tert-butyl--phenylnitrone (PBN) and -(4-pyridyl-1-oxyl)-N-tert-butylnitrone (4-POBN). When hypochlorite was added to (CH₃)₃COOH in the presence of a spin trap, an EPR spectrum appeared representing a superposition of two signals. One of them belonged to a spin adduct formed as a result of direct interaction of hypochlorite with the spin trap (hyperfine splitting constants were: _H H = 0.148 mT; a_N = 1.537 mT; and H_PP = 0.042 mT for 4-POBN and _H = 0.190 mT; a_N = 1.558 mT; and H_PP = 0.074 mT for PBN). The other signal was produced by hypochlorite interactions with (CH3)3COOH itself (hyperfine splitting constants were: _H = 0.233 mT; aN = 1.484 mT; H_PP = 0.063 mT and _H = 0.360 mT; a_N = 1.547 mT; H_PP = 0.063 mT for 4-POBN and PBN, respectively). Comparison of spectral characteristics of this spin adduct with those of tert-butoxyl or tert-butyl peroxyl radicals produced in known reactions of (CH₃)₃COOH with Fe²⁺ and Ce⁴⁺, respectively, showed that the radical (CH₃)₃COO. is produced from the interaction of hypochlorite with (CH₃)₃COOH^. Like Ce⁴⁺ but not Fe²⁺, hypochlorite addition to (CH₃)₃COOH was accompanied by a bright flash of chemiluminescence characteristic of the reactions in which peroxyl radicals are produced. Thus, all these results suggest peroxyl radical production in the reaction of hypochlorite with hydroperoxide. This reaction is one of the most possible ways for the initiation of free-radical LP that occurs in vivo, when hypochlorite interacts with unsaturated lipids comprising natural protein–lipid complexes, such as lipoproteins and biological membranes.     

Antioxidant activity of carotenoids: An electron-spin resonance study on β-carotene and lutein interaction with free radicals generated in a chemical system

β-Carotene is thought to be a chain-breaking antioxidant, even though we have no information about the mechanism of its antioxidant activity. Using electron-spin resonance (ESR) spectroscopy coupled to the spin-trapping technique, we have studied the effect of β-carotene and lutein on the radical adducts of the spin-trap PBN (N-t -butyl-α-phenylnitrone) generated by the metal-ion breakdown of different tert -butyl hydroperoxide (t BOOH) concentrations in methylene chloride. The peroxyl radical, along with an oxidation product of PBN (the PBNOx), trapped at room temperature from the breakdown of high concentration of t BOOH (1 M), were quenched by β-carotene or lutein, in competition with the spin-trapping agent. However, carotenoids were not able to quench the alkoxyl and methyl radicals generated in the reaction carried out in the presence of low t BOOH concentration (1 mM). The reaction between carotenoids and the peroxyl radical was also carried out in the absence of the spin trap, at 77 K: Under these different experimental conditions, we did not detect any radical species deriving from carotenoids. In the same system, a further evidence of the peroxyl radical quenching by β-carotene and lutein was obtained. The antioxidant activity of vitamin E was also tested, for comparison with the carotenoids. In the presence of α-tocopherol, peroxyl and alkoxyl radicals were quenched, and the tocopheroxyl radical was detected. Our data provide the first direct evidence that carotenoids quench peroxyl radicals. Under our experimental conditions, we did not detect any carotenoid radical species that could derive from the interaction with the peroxyl radical. The radical-trapping activity of β-carotene and lutein demonstrated in this chemical reaction contributes to our understanding carotenoid antioxidant action in biological systems. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 299–304, 1998     

Fluorescence resonance energy transfer for investigation of the interaction of Para Red with serum albumins

Para Red (PR) has been isolated from food additives, and shown to be toxic to humans. To facilitate examination of its toxicity, the interaction between PR and serum albumins (SA) was studied using fluorescence quenching and circular dichroism (CD) spectrophotometry. The experiments showed that the fluorescence intensity of serum albumins decreased with increasing concentrations of PR, which resulted from the binding of PR and SA. The binding constant, number of binding sites and thermodynamic parameters were calculated and hydrogen bond and van der Waals interactions were shown to play a key role in the binding process. Competition experiments indicated that PR mainly binds to Trp residues of SA within the site I. As the CD and three‐dimensional spectra revealed, the addition of PR induced a conformational change in SA. Copyright © 2015 John Wiley & Sons, Ltd.     

A comparative spin trapping study of the interaction of hypobromous and hypochlorous acids with <Emphasis Type="Italic">tert</Emphasis>-butyl hydroperoxide

Hypochlorite (HOCl/OCl^?) and hypobromite (HOBr/OBr^?) are shown to react with tert-butyl hydroperoxide with close rate constants (10.8 and 8.9 M^?1 s^?1, respectively). Using a spin trap α-(4-pyridyl-1-oxide)-N-tert-butyl nitrone, both reactions are shown to proceed through decomposition of the hydroperoxide yielding butylperoxyl [˙OOC(CH₃)₃] and butoxyl [˙OC(CH₃)₃] radicals in a ratio depending on the hydroperoxide concentration. Thus, like hypochlorite, hypobromite can generate free radicals in reactions with organic hydroperoxides, which can be important for intensification of free-radical processes, e.g., lipid peroxidation at the chain branching stage.     

A novel and simple method of screening compounds for interaction with DNA: A validation study

We report the development of a simple, cost-effective assay for detecting compounds that have the ability to interact with and modify DNA. Potential uses for the assay lie in the areas of early genotoxicity testing of drug candidates, anticancer and antibiotic drug discovery, environmental monitoring and testing in the food, beverage and cosmetics industries. At present the assay has been used to assess direct-acting compounds only and it is yet to be established whether the assay is compatible with bio-activation. The methodology is based on the oxidative reaction of potassium permanganate with pyrimidine bases, which have become perturbed and more reactive by the agent under test. Results are recorded by use of UV/vis spectroscopy. The adaptation to a multi-well plate format provides the capacity for high throughput utilizing small amounts of compounds. Over 100 compounds, comprising different classes of DNA-binding chemicals as well as non-binding controls, have been put through the assay and the results compared with existing genotoxicity testing data from other methods. The assay has shown to be predictive of the results of other genotoxicity testing methods. We have found that the method is overall predictive of 71% of Ames bacterial reverse-mutation test results (where data are given) encompassing both negative and positive results.     

A new methodology for compiling national Red Lists applied to butterflies (Lepidoptera, Rhopalocera) in Flanders (N-Belgium) and the Netherlands

The compilation of the Red Lists of butterflies in Flanders and the Netherlands was based on two criteria: a trend criterion (degree of decline) and a rarity criterion (actual distribution area). However, due to the large difference in mapping intensity in the two compared periods, a straightforward comparison of the number of grid cells in which each species was recorded, appeared inappropriate. To correct for mapping intensity we used reference species that are homogeneously distributed over the country, that have always been fairly common and that did not fluctuate in abundance too much during this century. For all resident species a relative presence in two compared periods was calculated, using the average number of grid cells in which these reference species were recorded as a correction factor. The use of a standardized method and well-defined quantitative criteria makes national Red Lists more objective and easier to re-evaluate in the future and facilitates the comparison of Red Lists among countries and among different organisms. The technique applied to correct for mapping intensity could be useful to other organisms when there is a large difference in mapping intensity between two periods.     

Isolation and the characterization of the degradation products of the mediator ABTS-derived radicals formed upon reaction with polyphenols

Two degradation products were obtained from the incubation of the widely used 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), ABTS, radical cations with the polyphenols, (+)-catechin, (-)-epicatechin, and phloroglucinol in acetate buffer (pH 5). The products were purified by reversed-phase chromatography and characterized by UV-visible detection, mass spectrometry, and (1)H NMR spectroscopy. The data allowed us to identify the degradation products as 3-ethyl-6-sulfonate benzothiazolinone imine and the corresponding sulfoxide, 3-ethyl-6-sulfonate benzothiazolone. Elemental composition strongly supported the proposed structures. Our results unequivocally demonstrated that ABTS radicals are not as stable as usually claimed because they could be degraded upon interaction with polyphenols, in addition to being reduced by these antioxidants back to the parent compound. Therefore, it is concluded that caution must be exercised in using ABTS radicals as a basis for the evaluation of antioxidant capacities of pure compounds and/or complex mixtures.     

Lifetime of peroxyl radicals of poly(U), poly(A) and single-and double-stranded DNA and the rate of their reaction with thiols

Peroxyl radicals of poly(U), poly(A), and single- and double-stranded DNA have been produced by photolysing H2O2 in oxygenated aqueous solution in presence of the substrates. The peroxyl radicals are formed by the reaction of OH radicals with the polynucleotides followed by addition of oxygen. The lifetime of the peroxyl radicals and the rate constant of their reactions with the thiols cysteamine, glutathione and dithiothreithol have been measured by time-resolved e.s.r. spectroscopy. The unusually long lifetimes range from 0.2 to 3.3 s. The activation energy for the decay for all four substrates is 10.3 +/- 1 kcal/mol (43 kJ mol-1). The reaction rate constants with the thiols range from k = 0.8 X 10(4) to 1.3 X 10(5) dm3 mol-1 s-1. The reactions of the thiols with the peroxyl radical of poly(U) are known to prevent strand break formation. This shows that the peroxyl radicals of poly(U) observed by e.s.r. are intermediates in the pathway leading to strand break formation.     

维生素E和硒水平对肉鸡不同自由基代谢的影响

选用200羽14日龄健康AA肉鸡,以电子自旋共振(ESR)捕集法和生物化学法对肉仔鸡血液和组织器官的不同自由基进行直接或间接测定,探讨VE和Se对肉鸡不同自由基代谢的作用及其动态变化.结果表明:组织一氧化氮(NO)自由基水平随日粮VE含量升高而降低,二者呈负相关关系,日粮高水平Se有诱导产生NO自由基的倾向;高VE和Se日粮显著提高血清和肝脏中SOD和GSH-Px的活性,但随处理时间的延长,组织SOD活性逐渐降低,而GSH-Px活性逐渐升高,说明日粮VE和/或Se不足均会诱导机体产生O.2-、H2O2自由基,且O2.-自由基会持续大量产生,而H2O2自由基仅在缺乏初期大量产生,而后趋于缓和;低VE和/或低Se均显著提高组织MDA含量,且低Se比低VE更为显著.VE和Se对肉鸡NO、O.2-和H2O2自由基代谢的作用存在协同效应.     

青葙红色素提取及稳定性研究

本文对青葙红色素的提取条件及稳定性进行了研究.结果表明:提取最佳条件以pH=3体积分数70%的乙醇为提取剂,物料比1 g:20mL,在35℃下浸提4 h,青葙花红色素对光、热稳定性较差,抗氧化性较好,在酸性介质中稳定.     

A simple and efficient synthesis of novel inhibitors of alpha-glucosidase based on benzimidazole skeleton and molecular docking studies

A novel series of benzimidazole derivatives were prepared starting from o-phenylenediamine and 4-nitro-o-phenylenediamine with iminoester hydrochlorides. Acidic proton in benzimidazole was exchanged with ethyl bromoacetate, then ethyl ester group was transformed into hydrazide group. Cyclization using CS₂/KOH leads to the corresponding 1,3,4-oxadiazole derivative, which was treated with phenyl isothiocyanate resulted in carbothioamide group, respectively. As the target compounds, triazole derivative was obtained under basic condition and thiadiazole derivative was obtained under acidic condition from cyclization of carbothioamide group. Most reactions were conducted using both the microwave and conventional methods to compare yields and reaction times. All compounds obtained in this study were investigated for α-glucosidase inhibitor activity. Compounds 6a, 8a, 4b, 5b, 6b and 7b were potent inhibitors with IC₅₀ values ranging from 10.49 to 158.2 μM. This has described a new class of α-glucosidase inhibitors. Molecular docking studies were done for all compounds to identify important binding modes responsible for inhibition activity of α-glucosidase.     

A novel method to predict protein-protein interactions based on the information of protein-protein interaction networks and protein sequence

Protein-protein interactions (PPIs) are crucial to most biochemical processes in human beings. Although many human PPIs have been identified by experiments, the number is still limited compared to the available protein sequences of human organisms. Recently, many computational methods have been proposed to facilitate the recognition of novel human PPIs. However the existing methods only concentrated on the information of individual PPI, while the systematic characteristic of protein-protein interaction networks (PINs) was ignored. In this study, a new method was proposed by combining the global information of PINs and protein sequence information. Random forest (RF) algorithm was implemented to develop the prediction model, and a high accuracy of 91.88% was obtained. Furthermore, the RF model was tested using three independent datasets with good performances, suggesting that our method is a useful tool for identification of PPIs and investigation into PINs as well.     

Detection of radicals in single droplets of oil-in-water emulsions with the lipophilic fluorescent probe BODIPY665/676 and confocal laser scanning microscopy

Lipid oxidation is a widespread phenomenon in foods and other systems of biological origin. Detection methods for early stages of lipid oxidation are in demand to understand the progress of oxidation in space and time. The fluorescence spectrum of the nonpolar fluorescent probe BODIPY^665/676 changes upon reacting with peroxyl radicals originating from 2,2′-azobis(2,4-dimethyl)valeronitrile and tert-butoxyl radicals generated from di-tert-butylperoxide. The excitation wavelength of the main peak of BODIPY^665/676 was 675 nm in the fluorometer, and 670 nm under the microscope, and the optimum excitation wavelength for the secondary peak of BODIPY^665/676 was 580 nm. Advantages of using BODIPY^665/676 are fewer problems with autofluorescence and the possibility of combining several fluorescent probes that are excited and emitted at lower wavelengths. However, because of the spectrum of the probe, specific lasers and detectors are needed for optimal imaging under the microscope. Furthermore, BODIPY^665/676 is resistant to photobleaching at both excitation wavelengths, 670 and 580 nm. In diffusion studies, BODIPY^665/676 is highly lipophilic, remaining in the lipid phase and not diffusing into the aqueous phase or between lipid droplets.     

Flexistipes sinusarabici,a novel genus and species of eubacteria occurring in the Atlantis II Deep brines of the Red Sea

Four isolates of a gram-negative flexible bacterium have been obtained from brine water samples of the Atlantis II Deep of the Red Sea at a depth of 2000 m. One isolate (MAS 10) was studied in detail. Cells are nonmotile, flexible rods, measuring about 0.3 m in width and 5 to 50 m in length. The new organisms are heterotrophs growing anaerobically on yeast extract, meat extract, peptone, tryptone, and, less efficiently, on acetate and casamino acids. Growth occurs between 30% and 53°C at pH 6 to 8 in the presence of at least 3% NaCl. The shortest doubling time is 8.5 h under optimal growth conditions. Cells are sensitive to the antibiotics penicillin, ampicillin, vancomycin, and streptomycin, but resistant to tetracyclin and rifamipicin. The GC-content of the DNA is 39 mol%. Based on their 16S rRNA the new isolates group with the general cluster of eubacterial phyla. Since they show no specific relationship to any of them, a new genus is described, which is named Flexistipes, the flexible stick. Type species is Flexistipes sinusarabici strain MAS 10 (DSM 4947).