CW EPR and 9 GHz EPR imaging investigation of stable paramagnetic species and their antioxidant activities in dry shiitake mushroom (Lentinus edodes)

We investigated the antioxidant activities and locations of stable paramagnetic species in dry (or drying) shiitake mushroom (Lentinus edodes) using continuous wave (CW) electron paramagnetic resonance (EPR) and 9?GHz EPR imaging. CW 9?GHz EPR detected paramagnetic species (peak-to-peak linewidth (ΔH_pp)?=?0.57?mT) in the mushroom. Two-dimensional imaging of the sharp line using a 9?GHz EPR imager showed that the species were located in the cap and shortened stem portions of the mushroom. No other location of the species was found in the mushroom. However, radical locations and concentrations varied along the cap of the mushroom. The 9?GHz EPR imaging determined the exact location of stable paramagnetic species in the shiitake mushroom. Distilled water extracts of the pigmented cap surface and the inner cap of the mushroom showed similar antioxidant activities that reduced an aqueous solution of 0.1?mM 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl. The present results suggest that the antioxidant activities of the edible mushroom extracts are much weaker than those of ascorbic acid. Thus, CW EPR and EPR imaging revealed the location and distribution of stable paramagnetic species and the antioxidant activities in the shiitake mushroom for the first time.     

EPR imaging and HPLC characterization of the pigment-based organic free radical in black soybean seeds

We investigated the location and distribution of paramagnetic species in dry black, brown, and yellow (normal) soybean seeds using electron paramagnetic resonance (EPR), X-band (9?GHz) EPR imaging (EPRI), and HPLC. EPR primarily detected two paramagnetic species in black soybean. These two different radical species were assigned as stable organic radical and Mn^2+?species based on the g values and hyperfine structures. The signal from the stable radical was noted at g?≈?2.00 and was relatively strong and stable. Subsequent noninvasive two-dimensional (2D) EPRI of the radical present in black soybean revealed that the stable radical was primarily located in the pigmented region of the soybean coat, with very few radicals observed in the soybean cotyledon (interior). Pigments extracted from black soybean were analyzed using HPLC. The major compound was found to be cyanidin-3-glucoside. Multi-EPR and HPLC results indicate that the stable radical was only found within the pigmented region of the soybean coat, and it could be cyanidin-3-glucoside or an oxidative decomposition product.     

Identification of a radical formed in the reaction mixture of rat brain homogenate with a ferrous ion/ascorbic acid system using HPLC–EPR and HPLC–EPR–MS

Identification of a free radical is performed for the reaction mixture of rat brain homogenate with a ferrous ion/ascorbic acid system using EPR, high performance liquid chromatography–electron paramagnetic resonance spectrometry (HPLC–EPR) and high performance liquid chromatography–electron paramagnetic resonance–mass spectrometry (HPLC–EPR–MS). EPR measurements of the reaction mixtures showed prominent signals with hyperfine coupling constants (α^N = 1.58 mT and α^Hβ = 0.26 mT). No EPR spectrum was detectable without rat brain homogenate, suggesting that the radical is derived from rat brain homogenate. An HPLC–EPR analysis of the reaction mixture showed a peak with retention time of 33.7 min. An HPLC–EPR–MS analysis of the peak gave two ions at m/z 224 and 137, suggesting that α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN)/ethyl radical adduct forms in the reaction mixture.     

Studies on the antioxidant activities of some new chromone compounds

Recent reviews evidence that the naturally occurring compounds containing the chromone skeleton exhibit antiradical activities, providing protection against oxidative stress. The antioxidant activities of 13 new synthesized chromonyl‐2,4‐thiazolidinediones, chromonyl‐2,4‐imidazolidinediones and chromonyl‐2‐thioxoimidzolidine‐4‐ones were evaluated using in vitro antioxidant assays, including superoxide anion radical (), hydroxyl radical (), 2,2‐diphenyl‐1‐picryl‐hydrazyl free radical (DPPH^?) scavenging capacity and total antioxidant capacity ferric ion reducing activity. Superoxide anion radical was produced using potassium superoxide/18‐crown‐6‐ether dissolved in dimethylsulfoxide, and the Fenton‐like reaction (Fe(II) + H₂O₂) was a generator of hydroxyl radicals. Chemiluminescence, spectrophotometry, electron paramagnetic resonance (EPR) and 5,5‐dimethyl‐1‐pyrroline‐N‐oxide (DMPO) as the spin trap were the measurement techniques. The results showed that the majority of the chromone derivatives tested showed a strong scavenging effect towards free radicals, similar to the chemiluminescence reaction with superoxide anion radical with a high activity, inhibition of the DMPO‐OOH radical EPR signal (24–58%), the DMPO‐OH radical EPR signal (4–75%) and DPPH radical EPR signal (6–100%) at 1 mmol/L. Several of the examined compounds exhibited the high reduction potentials. The results obtained show that the new synthesized chromone derivatives may directly scavenger reactive oxygen species and thus may play a protective role against oxidative damage. Copyright © 2014 John Wiley & Sons, Ltd.     

In vivo imaging of free radicals: Applications from mouse to man

Free radicals and other paramagnetic species, play an important role in cellular injury and pathophysiology. EPR spectroscopy and imaging has emerged as an important tool for non-invasive in vivo measurement and spatial mapping of free radicals in biological tissues. Extensive applications have been performed in small animals such as mice and recently applications in humans have been performed. Spatial EPR imaging enables 3D mapping of the distribution of a given free radical while spectral-spatial EPR imaging enables mapping of the spectral information at each spatial position, and, from the observed line width, the localized tissue oxygenation can be determined. A variety of spatial, and spectral-spatial EPR imaging applications have been performed. These techniques, along with the use of biocompatible paramagnetic probes including particulate suspensions and soluble nitroxide radicals, enable spatial imaging of the redox state and oxygenation in a variety of biomedical applications. With spectral-spatial EPR imaging, oxygenation was mapped within the gastrointestinal (GI) tract of living mice, enabling measurement of the oxygen gradient from the proximal to the distal GI tract. Using spatial EPR imaging, the distribution and metabolism of nitroxide radicals within the major organs of the body of living mice was visualized and anatomically co-registered by proton MRI enabling in vivo mapping of the redox state and radical clearance. EPR imaging techniques have also been applied to non-invasively measure the distribution and metabolism of topically applied nitroxide redox probes in humans, providing information regarding the penetration of the label through the skin and measurement of its redox clearance. Thus, EPR spectroscopy and imaging has provided important information in a variety of applications ranging from small animal models of disease to topical measurement of redox state in humans.     

Free radical scavenging activity of novel thiazolidine‐2,4‐dione derivatives

Free radical activity towards superoxide anion radical (), hydroxyl radical (HO^?) and 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH^?) of a series of novel thiazolidine‐2,4‐dione derivatives (TSs) was examined using chemiluminescence, electron paramagnetic resonance (EPR) and EPR spin trapping techniques. 5,5‐Dimethyl‐1‐pyrroline‐N‐oxide (DMPO) was applied as the spin trap. Superoxide radical was produced in the potassium superoxide/18‐crown‐6 ether dissolved in dimethyl sulfoxide. Hydroxyl radical was generated in the Fenton reaction (Fe(II) + H₂O₂. It was found that TSs showed a slight scavenging effect (15–38% reduction at 2.5 mmol/L concentration) of the DPPH radical and a high scavenging effect of (41–88%). The tested compounds showed inhibition of HO^? ‐dependent DMPO‐OH spin adduct formation (the amplitude of EPR signal decrease ranged from 20 to 76% at 2.5 mmol/L concentration. Our findings present new group compounds of relatively high reactivity towards free radicals. Copyright © 2012 John Wiley & Sons, Ltd.     

Evidence of in vivo Free Radical Generation by Spin Trapping with α-Phenyl N-Tert-Butyl Nitrone During Ischemia/Reperfusion in Rabbit Kidneys

By using α-phenyl N-tert-butyl nitrone (PBN) as spin trap molecule and the electron paramagnetic resonance (EPR) technique, we obtained the first direct evidence of in vivo intervention of free radicals during an ischemia (50 minutes) reperfusion phenomenon in kidney of an intact rabbit.

An EPR signal (triplet of doublets) characterized by coupling constants a_N = 14.75–15 G and a^H_s = 2.5–3 G was detected in blood samples. The signal was consistent with a nitroxyl-radical adduct resulting from the spin trapping by PBN of either oxygen-or carbon-centered radicals. Control experiments indicated that the EPR signal was not due to a toxic effect of the spin trap molecule.     

Towards in vivo melanin radicals detection in melanomas by electron paramagnetic resonance (EPR) spectroscopy: a proof-of-concept study

Melanoma is the most aggressive skin tumour type. Although complete cure can be achieved when the whole tumour is resected, prognostic dramatically drops when melanoma cells reach deeper tissues and lymph nodes. Hence, there is an urgent need to develop accurate tools allowing (i) discriminating benign naevi from malignant tumours and (ii) being able to characterise melanoma infiltration. For that purpose, we exploited the paramagnetic properties of melanin by using electron paramagnetic resonance (EPR) spectroscopy to measure the melanin content in pigmented (B16F10 cancer cells) and non-pigmented melanomas (WM2664 cancer cells) inoculated intradermally in nude mice. Specifically, we took advantage of a new clinical EPR device (1?GHz), which provides sensitive measurements of radical species in vivo. Results showed that the melanin-specific EPR signal increased with tumour growth in pigmented tumours, whereas no EPR signal could be detected in achromic melanomas. These data plead for the development of new EPR spectrometers/imagers with an improved in-depth resolution for the detection of invasive melanomas.     

The Fate of Mn2+ Ions Inside Saccharomyces cerevisiae Cells Seen by Electron Paramagnetic Resonance

The fate of Mn²⁺ inside Saccharomyces cerevisiae cells was monitored during import and export processes, using information from electron paramagnetic resonance (EPR) spectroscopy analysis. EPR spectra showed that when entering the cell in high number, manganese ions immediately precipitated. If recorded under conditions that favored manganese efflux, EPR spectra indicated that only osmotically free ions were exported and that bound manganese had to turn into the soluble form before being able to leave the cell.     

Detection and identification of oxidants formed during •NO/O2•– reaction: A multi-well plate CW-EPR spectroscopy combined with HPLC analyses

Abstract

New techniques and probes are routinely emerging for detecting short-lived free radicals such as superoxide radical anion (O₂^?–), nitric oxide (^?NO), and transient oxidants derived from peroxynitrite (ONOO^–/ONOOH). Recently, we reported the profiles of oxidation products (2-hydroxyethidium, ethidium, and various dimeric products) of the fluorogenic probe hydroethidine (HE) in the ^?NO/O₂^?– system (Zielonka et al. 2012). In this study, we used HPLC analyses of HE oxidation products in combination with continuous wave electron paramagnetic resonance (CW-EPR) spin trapping with 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) to define the identity of the oxidizing species formed in the ^?NO/O₂^?– system. EPR spin-trapping technique is still considered as the gold standard for characterization of free radicals and their intermediates. We monitored formation of BMPO-superoxide (BMPO-^?OOH) and BMPO-hydroxyl (BMPO-^?OH) radical adducts. Simultaneous analyses of results from EPR spin-trapping and HPLC measurements are helpful in the interpretation of the mechanism of formation of products of HE oxidation.     

Antioxidant Capacity and Chemical Profiles of Satureja montana L. Honey: Hotrienol and Syringyl Derivatives as Biomarkers

The present study is focused on the antioxidant capacity and chemical profiling of eight Croatian Satureja montana L. honey samples. Among the 20 compounds obtained by headspace solid‐phase microextraction (HS‐SPME) and identified by GC‐FID and GC/MS analyses, hotrienol was predominant (75.9–81.7%). The honey matrix volatile/semivolatile profile was investigated by ultrasonic solvent extraction (USE) followed by GC‐FID and GC/MS analyses. The major compounds identified by this latter method were the sinapic‐acid derivatives methyl syringate (36.2–72.8%) and syringaldehyde (2.2–43.1%). Direct, targeted HPLC‐DAD analyses of the native honey samples revealed the presence of methyl syringate (7.10–39.60 mg/kg) and syringic acid (0.10–1.70 mg/kg). In addition, the total phenolic content of the samples was determined by the Folin? Ciocalteu assay (311.0–465.9 mg GAE/kg), and the antioxidant capacity was evaluated by the DPPH radical‐scavenging activity (0.5–1.0 mmol TEAC/kg) and the ferric reducing antioxidant power (2.5–5.1 mmol Fe²⁺/kg).     

Triarylmethyl-based biradical as a superoxide probe

Abstract

Superoxide radical represents one of the most biologically relevant reactive oxygen species involved in numerous physiological and pathophysiological processes. Superoxide measurement through the decay of an electron paramagnetic resonance (EPR) signal of a triarylmethyl (TAM) radical possesses the advantage of a high selectivity and relatively high rate constant of TAM reaction with the superoxide. Hereby we report a straightforward synthesis and characterization of a TAM–TAM biradical showing a high reactivity with superoxide (second-order rate constant, (6.7?±?0.2)?×?10³ M^?1 s^?1) enabling the measurement of superoxide radical by following the increase of a sharp EPR signal associated with the formation of a TAM-quinone-methide monoradical product.     

Mössbauer spectroscopy and electron paramagnetic resonance studies of iron metabolites inPseudomonas aeruginosa: Fe2+ and Fe3+ ferritin in57ferripyoverdine incubated cells and57ferric citrate fed cells

Pseudomonas aeruginosa samples were studied using Mössbauer spectroscopy and electron paramagnetic resonance (EPR). Samples included whole cells, membranes, and soluble fractions from cells which had been grown with⁵⁷ferric chloride,⁵⁷ferric citrate or incubated with⁵⁷ferripyoverdine. These experiments show for the first time thatP. aeruginosa can accumulate iron in a bacterioferritin when grown under conditions of iron limitation and incubated with its cognate ferrisiderophore, ferripyoverdine. Soluble fraction fromP. aeruginosa cells which were grown iron starved and incubated with⁵⁷ferripyoverdine for 120 min showed the presence of both a ferric and ferrous complex whose Mössbauer spectra matched that of bacterioferritin extracted fromAzotobacter vinelandii and whose EPR spectra showed a characteristic ferritin-like resonance. A second soluble fraction sample from cells which had been grown with⁵⁷ferric citrate also showed the presence of a species with the same EPR and Mössbauer parameters. In addition Western blotting confirmed the presence of bacterioferritin in the soluble fraction of the cells which had been incubated with ferripyoverdine.     

In vitro Biological Activities of Seed Essential Oils from the Cameroonian Spices Afrostyrax lepidophyllus Mildbr. and Scorodophloeus zenkeri Harms Rich in Sulfur‐Containing Compounds

The chemical composition of the essential oils obtained from the seeds of bush onion (Afrostyrax lepidophyllus) and tropical garlic tree (Scorodophloeus zenkeri), plants used as spices in the traditional African cuisine, was determined by GC‐FID and GC/MS analyses. Moreover, in vitro biological properties of the oils, namely, the cytotoxic, antioxidant, and antimicrobial activities, were investigated by the MTT, the DPPH^. and ABTS^.+ scavenging, and the agar disc‐diffusion methods, respectively. Both oils were composed mainly by S‐containing compounds, accounting for 91.0–96.1% of the total oil compositions, which provided them the typical garlic‐ and onion‐like odors of spices. The predominant compound in both oils, 2,4,5,7‐tetrathiaoctane ( 1 ; 51.5–52.9%), was isolated by preparative TLC and structurally elucidated by ¹H‐ and ¹³C‐NMR data. The oils exhibited a strong inhibitory effect on the growth of human cancer cells, namely, T98G (human glioblastoma multiforme cell line), MDA‐MB 231 (human breast adenocarcinoma cell line), A375 (human malignant melanoma cell line), and HCT116 (human colon carcinoma cell line) cells, and a good DPPH^.‐ and ABTS^.+‐scavenging activity, while the antimicrobial effects were negligible. The volatile compositions of A. lepidophyllus and S. zenkeri oils supported their use as odorous spices. The significant inhibition activities detected make these oils worthy of further investigation as promising chemopreventive agents to be exploited in the African pharmaceutical market.     

Alterations in metabolite profile and free radical scavenging activities of <Emphasis Type="Italic">Withania somnifera</Emphasis> leaf and root extracts under supplemental ultraviolet-B radiation

The effects of supplemental ultraviolet-B (s-UV-B; 3.6 kJ m^?2 day^?1 above ambient) radiation on plant metabolite profile and free radical scavenging activities of Withania somnifera (an indigenous medicinal plant) under field conditions were investigated. The metabolite profiles of both leaves and roots were analyzed via GC–MS. The methanolic extracts were examined for their DPPH radical-, superoxide radical-, hydrogen peroxide-, hydroxyl radical-, and nitric oxide radical scavenging activities, ferrous ion chelating activity, and reducing power. GC–MS profile of leaves revealed increment in compounds such as isophytol (138.1 %), β-stigmasterol (183.05 %), trans-squalene (233.3 %), and Withaferin A (155.0 %), while compounds such as eugenol, β-carotene, lycopene, and vitamin E were detected in s-UV-B-treated leaves only. In roots, compounds such as ledol, neophytadiene, palmitic acid, retinol, sitosteryl oleate, and campesterol registered their presence only under s-UV-B. Methanolic extracts of treated plant organs were found to be more potent as free radical scavengers (their EC₅₀ values being lower than those of control extracts). Anomalies were observed for nitric oxide radical scavenging in both leaves and roots. The present study indicates that s-UV-B alters the composition and contents of plant metabolites leading to an increase in their free radical scavenging activities. Hence, s-UV-B-treated plant organs might be more effective in combating oxidative stress as well as from a nutritional and health perspective.     

Stable free radicals in ozone-damaged wheat leaves

Chlorophyll fluorescence measurements were performed on attached leaves of wheat plants (Triticum aestivum L. cv. Nandu) that were exposed to ambient air and to air supplemented with 80 and 120nmol mol^-1 ozone. Decreases in the “current photochemical capacity” were observed that were dependent on both the ozone concentration and duration of exposure. Electron paramagnetic resonance (EPR) spectra on freeze-dried samples from the same batches of plants showed the presence of an unidentified stable free radical, whose spectra had similarities to that of the ubisemiquinone radical. The intensity of this radical signal increased with the duration of ozone exposure in leaves that received an additional 120nmol mol^-1 ozone. In contrast, with exposure to air with 80nmol mol^-1 added ozone, there was little if any change in free radical signal intensity over the 4 week period of the experiment. The increase in intensity of the EPR signal occurred later than the chlorophyll fluorescence changes, which suggests that it is associated with permanent leaf damage.     

Singlet oxygen scavenging activity of tocopherol and plastochromanol in Arabidopsis thaliana: relevance to photooxidative stress

In the present study, singlet oxygen (¹O₂) scavenging activity of tocopherol and plastochromanol was examined in tocopherol cyclase‐deficient mutant (vte1) of Arabidopsis thaliana lacking both tocopherol and plastochromanol. It is demonstrated here that suppression of tocopherol and plastochromanol synthesis in chloroplasts isolated from vte1 Arabidopsis plants enhanced ¹O₂ formation under high light illumination as monitored by electron paramagnetic resonance spin‐trapping spectroscopy. The exposure of vte1 Arabidopsis plants to high light resulted in the formation of secondary lipid peroxidation product malondialdehyde as determined by high‐pressure liquid chromatography. Furthermore, it is shown here that the imaging of ultra‐weak photon emission known to reflect oxidation of lipids was unambiguously higher in vte1 Arabidopsis plants. Our results indicate that tocopherol and plastochromanol act as efficient ¹O₂ scavengers and protect effectively lipids against photooxidative damage in Arabidopsis plants.     

A novel ascorbic acid-resistant nitroxide in fat emulsion is an efficient brain imaging probe for in vivo EPR imaging of mouse

Abstract

The loss of paramagnetism of nitroxide radicals due to reductant reactions in biological systems, places a fundamental time constraint on their application as an imaging probe in in vivo EPR imaging studies. However, in vitro studies of the newly synthesized tetraethyl-substituted piperidine nitroxide radical demonstrated high resistivity to paramagnetic reduction when exposed to ascorbic acid, a common reduction agent in biological systems. In this work we investigated the use of these nitroxides as an imaging probe in EPR imaging of small rodents. 2,2,6,6-Tetraethyl-piperidine nitroxide (TEEPONE) is not highly soluble in aqueous media, thus a lipid-based emulsion system of lecithin was used to solubilize TEEPONE. The obtained solution was homogenous and with low viscosity, allowing smooth intravenous injection into mice tail vein. Acquired three dimensional (3D) EPR images of mouse head clearly showed TEEPONE distributed in all tissues including brain tissues, with an average measurable signal half-life of more than 80 min, thus demonstrating high resistivity to reduction due to ascorbic acid in in vivo animal studies, and the potential for use of this compound in in vivo studies of animal model systems.     

EPR Spin-Trapping and Spin-Probing Spectroscopy in Assessing Antioxidant Properties: Example on Extracts of Catkin, Leaves, and Spiny Burs of Castanea sativa

Electron paramagnetic resonance (EPR) spin-trapping and spin-probing techniques were applied to determine antioxidant activity of extracts of catkin, leaves, and spiny burs of Castanea sativa against physiologically relevant reactive species—superoxide and hydroxyl radical generated in simple chemical systems and hydrogen peroxide applied on erythrocytes. Efflux of K⁺ was used as a marker of membrane integrity. Chemical composition of extracts was analyzed using HPLC/DAD and LC/MS. Extracts showed high antioxidative capacity against superoxide but lower activity against hydroxyl radical. They protected fluidity and integrity of membranes of erythrocytes exposed to hydrogen peroxide. Levels of derivatives of ellagitannins showed positive correlation with the antioxidative activity of extracts. Therefore, ellagitannins from chestnut extracts could represent easily accessible natural antioxidants and beneficial component of human diet in pathophysiological conditions related to oxidative stress. In conclusion, EPR spectroscopy represents a valuable tool for evaluation of antioxidant activity in both hydrophilic and lipophilic media. This work was supported by the Federal Ministry of Education and Science of Bosnia and Herzegovina Grant No. 614300 and the Ministry of Science, Technology, and Development of Republic of Serbia Grant No. 143016. We would like to thank Ms. Ana Martinović on technical support.     

Mitochondrial dysfunction in patients with severe sepsis: An EPR interrogation of individual respiratory chain components

Electron paramagnetic resonance (EPR) spectra of complex biological systems contain information about the paramagnetic centres present. Retrieving such information is important since paramagnetic species are common intermediates of all redox reactions in both normal and abnormal metabolism. However, it is often difficult to determine the nature and content of all paramagnetic species present because the EPR signals from individual centres overlap. Here, we apply our deconvolution method based on spectra subtraction with variable coefficient to quantify individual paramagnetic components of human muscle biopsies taken from critically ill patients with severe sepsis. We use low temperature EPR spectroscopy to identify and quantify nine different paramagnetic species in the tissue. These include the majority of the mitochondrial iron-sulfur centres and the first in vivo report of a mitochondrial radical assigned to a spin-coupled pair of semiquinones (SQ^·-SQ^·). We have previously demonstrated in these same muscle biopsies that biochemical assays of mitochondrial dysfunction correlate with clinical outcomes (D. Brealey, M. Brand, I. Hargreaves, S. Heales, J. Land, R. Smolenski, N.A. Davies, C.E. Cooper, M. Singer, Association between mitochondrial dysfunction and severity and outcome of septic shock. Lancet 360 (2002) 219-223.). Analysis of the paramagnetic centres in the muscle confirms and extends these findings: the (SQ^·-SQ^·) radical species negatively correlates with the illness severity of the patient (APACHE II score) and a decreased concentration of mitochondrial Complex I iron-sulfur redox centres is linked to mortality.