Free radical formation in the cochlea plays a key role in the development of noise-induced hearing loss (NIHL). The amount, distribution, and time course of free radical formation have been defined, including a clinically significant formation of both reactive oxygen species and reactive nitrogen species 7-10 days after noise exposure. Reduction in cochlear blood flow as a result of free radical formation has also been described. Here we report that the antioxidant agents vitamins A, C, and E act in synergy with magnesium to effectively prevent noise-induced trauma. Neither the antioxidant agents nor the magnesium reliably reduced NIHL or sensory cell death with the doses we used when these agents were delivered alone. In combination, however, they were highly effective in reducing both hearing loss and cell death even with treatment initiated just 1 h before noise exposure. This study supports roles for both free radical formation and noise-induced vasoconstriction in the onset and progression of NIHL. Identification of this safe and effective antioxidant intervention that attenuates NIHL provides a compelling rationale for human trials in which free radical scavengers are used to eliminate this single major cause of acquired hearing loss.

Tyrosinase enzyme plays a crucial role in melanin biosynthesis and enzymatic browning process of vegetables and fruits. A series of veratric acid derivatives containing benzylidene-hydrazine moieties with different substitutions were synthesized and their inhibitory effect on mushroom tyrosinase and free radical scavenging activity were evaluated. The results indicated that N'-(4-chlorobenzylidene)-3,4-dimethoxybenzohydrazide (D5) and N'-(2,3-dihydroxybenzylidene)-3,4-dimethoxybenzohydrazide (D12) showed the highest tyrosinase inhibitory activity with IC50 values of 19.72 ± 1.84 and 20.63 ± 0.79 μM, respectively, that were comparable with the IC50 value of kojic acid (19.08 ± 1.21 μM). D12 was also a potent radical scavenger with EC50 value of 0.0097 ± 0.0011 mM. The free radical scavenging activity of D12 was comparable with the standard quercetin. The inhibition kinetic analyzed by Lineweaver-Burk plots revealed that compound D5 was a competitive tyrosinase inhibitor. Molecular docking study was carried out for the derivatives demonstrating tyrosinase inhibitory activity. D5 and D12 possessed the most negative estimated free energies of binding in mushroom tyrosinase active site. Therefore, D5 and D12 could be introduced as potent tyrosinase inhibitors that might be promising leads in medicine, cosmetics and food industry.

Hydroxystilbenes are naturally occurring polyphenols with protective effects against reactive oxygen and nitrogen species (ROS/RNS). Here, we investigated oxyresveratrol (OXY), which is contained in high amounts in mulberry wood, in comparison to the antioxidant resveratrol (RES). We found that OXY is a more effective scavenger for 2,2-diphenyl-1-picryl-hydrazyl (DPPH, 100 microM) used as a general free radical model, compared to RES or trans-4-hydroxystilbene (IC(50)=28.9, 38.5, and 39.6 microM, respectively). When primary glial cell cultures were loaded with the ROS/RNS-sensitive fluorochrome 2,7-dichlorodihydrofluorescein, the lowest rise in the fluorescence signal after H(2)O(2) exposure was seen when the cells were pretreated with OXY. Using 4,5-diaminofluorescein (DAF-2) to monitor free nitric oxide levels (7.7 microM NO) in a spectrofluorimetric cell-free assay, we found again that OXY (at 5 microM) is a more effective scavenger. Accordingly, cultures of the murine microglial cell line N9 and primary mixed glial cultures were used to test the drug effects of NO production upon expression of the inducible isoform of nitric oxide synthase (iNOS). We found that both compounds considerably diminished NO (nitrite) levels, RES more effectively than OXY (IC(50)=22.36 and 45.31 microM). RES but not OXY down-regulated the expression of iNOS protein, but both did not alter iNOS activity. Furthermore, OXY displayed a generally lower cytotoxicity than RES. The radical and ROS scavenging properties, as well as the lower cytotoxicity towards microglia and the known good water solubility suggest OXY as a potential protectant against ROS/RNS.

5-Lipoxygenase (5-LO) is the key enzyme responsible for the conversion of arachidonic acid to leukotrienes, a class of lipid mediators implicated in inflammatory disorders. In this paper, we describe the design, synthesis, and preliminary activity studies of novel clicked caffeic esters and amides as radical scavengers and 5-LO inhibitors. From known 5-LO inhibitor 3 as a lead, cinnamic esters 8a-h and amides 9a-h as well as caffeic esters 15a-h and amides 16a-h were synthesized by Cu(I)-catalyzed [1,3]-dipolar cycloaddition with the appropriate azide precursors and terminal alkynes. All caffeic analogs are proved to be good radical scavengers (IC50: 10-20 μM). Esters 15g and 15f possessed excellent 5-LO inhibition activity in HEK293 cells and were equipotent with the known 5-LO inhibitor CAPE and more potent than Zileuton. Several synthesized esters possess activities rivaling Zileuton in stimulated human polymorphonuclear leukocytes.

Silibinin is the main biologically active component of silymarin extract and consists of a mixture 1:1 of two diastereoisomeric flavonolignans, namely silybin A (1a) and silybin B (1b), which we call here silybins. Despite the high interest in the activity of this flavonolignan, there are still few studies that give due attention to the role of its stereochemistry and, there is still today a strong need to investigate in this area. In this regard, here we report a study concerning the radical scavenger ability and the antiproliferative activity on different cell lines, both of silybins and phosphodiester-linked silybin dimers. An efficient synthetic strategy to obtain silybin dimers in an optical pure form (6aa, 6ab and 6bb) starting from a suitable building block of silybin A and silybin B, obtained by us from natural extract silibinin, was proposed. New dimers show strong antioxidant properties, determined through hydroxyl radical (HO●) scavenging ability, comparable to the value reported for known potent antioxidants such as quercetin. A preliminary screening was performed by treating cells with 10 and 50 μM concentrations for 48 h to identify the most sensitive cell lines. The results show that silibinin compounds were active on Jurkat, A375, WM266, and HeLa, but at the tested concentrations, they did not interfere with the growth of PANC, MCF-7, HDF or U87. In particular, both monomers (1a and 1b) and dimers (6aa, 6ab and 6bb) present selective anti-proliferative activity towards leukemia cells in the mid-micromolar range and are poorly active on normal cells. They exhibit different mechanisms of action in fact all the cells treated with the 1a and 1b go completely into apoptosis, whereas only part of the cells treated with 6aa and 6ab were found to be in apoptosis.

With the recent development of chemical analysis technology, attention has been placed on natural light-sensitive compounds that exhibit photoreactivity to expand the structural diversity of natural product chemistry. Photochemical reactions that proceed via a free radical mechanism could be used to modulate the radical-scavenging ability of natural products as well as involve structural change. As the health benefits of radicals are also presented, there is a need for a controllable radical scavenging method for topical and selective application. In this study, we developed a novel acquisition and processing method to identify light-controlled radical scavengers in plant extracts and evaluate their antioxidant activity under light irradiation based on in situ UV-LED NMR spectroscopy. Using the developed method, licochalcones A and B, in which the trans and cis isomers undergo reversible photoisomerization, were selectively identified from licorice root extract, and their light-induced free radical scavenging activity was confirmed.

Securigera varia (Fabaceae) is a common herbaceous perennial plant widely growing in Europe and Asia and purposely established for erosion control, roadside planting, and soil rehabilitation. The aim of this study was to determine the radical scavenging activity of a crude methanol extract of S. varia aerial parts by using the free radical DPPH (1,1-diphenyl-2-picrylhydrazyl) and to rapidly identify the compounds involved in this activity. The crude extract was initially separated in five fractions on Diaion HP20 resin and the most active part was fractionated by Centrifugal Partition Extraction (CPE). Known compounds were directly identified by a (13)C-NMR-based dereplication method. Semi-preparative high performance liquid chromatography purification experiments were further performed to identify unknown or minor active compounds. As a result, one new (13) and twelve known flavonoid glycosides together with three nitropropanoylglucopyranoses were isolated, including astragalin (1), kaempferol-3-O-(6-O-acetyl)-β-D-glucopyranoside (2), kaempferol-3,4'-di-O-β-D-glucopyranoside (3), trifolin (4), isoquercitrin (5), hyperoside (6), isovitexin (7), isoorientin (8), isovitexin 4'-O-β-D-glucopyranoside (9), apigenin 7-O-β-D-glucuronopyranoside (10), luteolin 7-O-β-D-glucuronopyranoside (11), apigenin 7-O-α-L-rhamnopyranosyl-(1→2)-β-D-glucuronopyranoside (12), apigenin 7-O-β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranoside (13), 6-O-(3-nitropropanoyl)-β-D-glucopyranoside (14), coronillin (16) and coronarian (15). 120 mg of the most active compound isoorientin against the free radical DPPH was recovered by CPE with an HPLC purity of 99%.

Oxygen and nitrogen derived molecules mediated oxidation and nitration have been involved in several pathological conditions. Conversely, nitric oxide and hydrogen peroxide are important signalization intermediates, whose concentrations are tightly regulated by specialized enzyme repertoires and should remain undisturbed by the addition of exogenous antioxidant molecules, as already demonstrated by intervention studies with antioxidant vitamins. Our goal was to develop specific antioxidants able to scavenge peroxynitrite anion, as well the radicals derived from the homolytic decomposition of its conjugated acid, nitrogen dioxide and hydroxyl radical. Fourteen substituted nitroalkenes, seven 4-substituted 1-(2-nitro-1Z-ethenyl)benzene, and seven 4-substituted (2-nitro-1Z-propenyl)benzene, with different stereochemical and electronic characteristics were synthesized and tested. Compounds with the electron donor group N,N-dimethylamino showed the highest reaction rates against peroxynitrite, and also reacted with its homolytic decomposition products, OH and NO₂. While 1,1-dimethylamino-4-(2-nitro-1Z-ethenyl)benzene came up as a lead for future developments without the risk of interfering with signalization pathways, since it was highly specific for peroxynitrite and peroxynitrite derived radicals, its methylated analogous 1,1-dimethylamino-4-(2-nitro-1Z-propenyl)benzene was less specific and also reacted with NO and O₂(-), the biological precursor of H₂O₂.

1. Poly (ADP-ribose) synthetase (PARS) is a nuclear enzyme activated by strand breaks in DNA which are caused by reactive oxygen species (ROS) and peroxynitrite. Excessive activation of PARS may contribute to the hepatocyte injury caused by ROS in vitro and inhibitors of PARS activity reduce the degree of reperfusion injury of the heart, skeletal muscle and brain in vivo. Here we compared the effects of various inhibitors of the activity of PARS with those of deferoxamine (an iron chelator which prevents the generation of hydroxyl radicals) and tiron (an intracellular scavenger of superoxide anion) on the degree of hepatic injury caused by ischaemia and reperfusion of the liver in the anaesthetized rat or rabbit. 2. In the rat, ischaemia (30 or 60 min) and reperfusion (120 min) of the liver resulted in significant increases in the serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) indicating the development of liver injury. Intravenous administration of the PARS inhibitors 3-aminobenzamide (3-AB, 10 mg kg(-1) or 30 mg kg(-1)), 1,5-dihydroxyisoquinoline (ISO, 1 mg kg(-1)) or 4-amino-1,8-naphthalimide (4-AN, 3 mg kg(-1)) before reperfusion did not reduce the degree of liver injury caused by ischaemia-reperfusion. 3. In contrast to the PARS inhibitors, deferoxamine (40 mg kg(-1)) or tiron (300 mg kg(-1)) significantly attenuated the rise in the serum levels of AST and ALT caused by ischaemia-reperfusion of the liver of the rat. 4. In the rabbit, the degree of liver injury caused by ischaemia (60 min) and reperfusion (120 min) was also not affected by 3-AB (10 mg kg(-1)) or ISO (1 mg kg(-1)). 5. These results support the view that the generation of oxygen-derived free radicals mediates the liver injury associated with reperfusion of the ischaemic liver by mechanism(s) which are independent of the activation of PARS.

Exposure to elevated levels of manganese has been shown to cause neuronal damage in the midbrain and the development of Parkinsonian symptoms. Activation of microglia and release of neurotoxic factors in particular free radicals are known to contribute to neurodegeneration. We have recently reported that manganese chloride (MnCl(2)) stimulates microglia to produce reactive oxygen species (ROS). The aim of this study is to determine the role of microglia in the MnCl(2)-induced degeneration of dopaminergic (DA) neurons that are particularly vulnerable to oxidative insult. MnCl(2) (10-300 microM; 7 days) was markedly more effective in damaging DA neurons in the rat mesencephalic neuron-glia cultures than the neuron-enriched (microglia-depleted) cultures. In addition, the microglia-enhanced MnCl(2) toxicity was found to be preferential to DA neurons. The microglial enhancement of DA neurotoxicity was further supported by the observation that replenishment of microglia to the neuron-enriched cultures significantly increased the susceptibility of DA neurons to the MnCl(2)-induced damage. Analysis of the temporal relationship between microglial activation and DA neurodegeneration revealed that MnCl(2)-stimulated microglial activation preceded DA neurodegeneration. Mechanistically, MnCl(2) (10-300 microM) stimulated a concentration- and time-dependent robust production of ROS and moderate production of nitric oxide but no detectable release of tumor necrosis factor-alpha and interleukin-1beta. Application of free radical scavengers including superoxide dismutase/catalase, glutathione, N-acetyl cysteine and an inhibitor of nitric oxide biosynthesis significantly protected DA neurons against the MnCl(2)-induced degeneration. These results demonstrate that microglial activation and the production of reactive nitrogen and oxygen free radicals promote the MnCl(2)-induced DA neurodegeneration.

In developing brain, we have previously shown both in vivo [L.D. Longo, S. Packianathan, J.A. McQueary, R.B. Stagg, C.V. Byus and C.D. Cain, Acute hypoxia increases ornithine decarboxylase activity and polyamine concentrations in fetal rat brain, Proc. Natl. Acad. Sci. USA, Vol. 90 (1993) 692-696] and in vitro [S. Packianathan, C.D. Cain, B.H. Liwnicz and L.D. Longo, Ornithine decarboxylase activity in vitro in response to acute hypoxia: a novel use of newborn rat brain slices, Brain Res., Vol. 688 (1995) 61-71] that acute hypoxia is associated with a significant increase in ornithine decarboxylase (ODC) activity and polyamine concentrations. We tested the hypothesis that oxygen free radicals induce an increase in ODC activity similar to that of hypoxia and that both this and the hypoxia-induced response are inhibited by free radical scavengers.

Retinal (C20) and the C25 and C30 homologues were compared as radical scavengers together with their C22, C27, and C32 homologues linked with daidzein through a B'3 (isoflavonoid) to oxo-carbon (aldehyde) covalent bond. Oxidation potential in acetonitrile determined by cyclic voltammetry and ionization potential calculated by density functional theory for the aldehydes and dyads (conjugates), of which the two longer are new, decreased linearly with the wavenumber for absorption maximum. The logarithm of the second-order rate constant for scavenging of the ABTS•+ increased linearly with decreasing oxidation potential suggesting that longer conjugation in the antioxidant increases the rate of electron transfer. A similar linear free energy relationship was found for the rate of scavenging DPPH•, including daidzein, which may indicate involvement of hydrogen atom transfer from an isoflavonoid phenol. Prediction of radical scavenging efficiency from visible absorption spectra was demonstrated with the perspective of rational design of bifunctional amphiphilic antioxidants.

To evaluate the antioxidant activity of potential synthetic enzyme mimetics, we prepared new five copper(II) complexes via a self-assembly method and named them [Cu(2-(HOCH2)py)3](ClO4)2 (1), [Cu(2-(HOCH2)py)2(H2O)2]SiF6 (2), [Cu2(2-(HOCH2CH2)py)2(2-(OCH2CH2)py)2](ClO4)2 (3), [Cu(pyBIm)3](BF4)2·1.5H2O (4) and [Cu(py2C(OH)2)2](ClO4)2 (5). The synthetic protocol involved N,O- or N,N-donors: 2-(hydroxymethyl)pyridine (2-(HOCH2)py), 2-(hydroxyethyl)pyridine (2-(HOCH2CH2)py), 2-(2-pyridyl)benzimidazole (pyBIm), di(2-pyridyl)ketone (py2CO). The obtained Cu(II) complexes were fully characterised by elemental analysis, FTIR, EPR, UV-Vis, single-crystal X-ray diffraction and Hirshfeld surface analysis. Crystallographic and spectroscopic analyses confirmed chromophores of both monomeric ({CuN3O3} (1), {CuN2O4} (2), {CuN6} (4), {CuN4O2} (5)) and dimeric complex ({CuN2O3} (3)). Most of the obtained species possessed a distorted octahedral environment, except dimer 3, which consisted of two copper centres with square pyramidal geometries. The water-soluble compounds (1, 3 and 5) were selected for biological testing. The results of the study revealed that complex 1 in solutions displayed better radical scavenging activity than complexes 3, 5 and free ligands. Therefore, complex 1 has been selected for further studies to test its activity as an enzyme mimetic. The chosen compound was tested on the erythrocyte lysate of two groups of patients after undergoing chemotherapy and chemoradiotherapy. The effect of the tested compound (1) on enzyme activity levels (TAS, SOD and CAT) suggests that the selected complex can be treated as a functional mimetic of the enzymes.

Hepatic ischemia-reperfusion injury (HIRI) is a pathophysiological process during liver transplantation, characterized by insufficient oxygen supply and subsequent restoration of blood flow leading to an overproduction of reactive oxygen species (ROS), which in turn activates the inflammatory response and leads to cellular damage. Therefore, reducing excess ROS production in the hepatic microenvironment would provide an effective way to mitigate oxidative stress injury and apoptosis during HIRI. Nanozymes with outstanding free radical scavenging activities have aroused great interest and enthusiasm in oxidative stress treatment.

Twenty-five piperidines were studied as potential radical scavengers and antitumor agents. Quantitative interaction of compounds with ctDNA using spectroscopic techniques was also evaluated. Our results demonstrate that the evaluated piperidines possesses different abilities to scavenge the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) and the anion radical superoxide (•O2-). The piperidine 19 was the most potent radical DPPH scavenger, while the most effective to •O2- scavenger was piperidine 10. In general, U251, MCF7, NCI/ADR-RES, NCI-H460 and HT29 cells were least sensitive to the tested compounds and all compounds were considerably more toxic to the studied cancer cell lines than to the normal cell line HaCaT. The binding mode of the compounds and ctDNA was preferably via intercalation. In addition, these results were confirmed based on theoretical studies. Finally, a linear and exponential correlation between interaction constant (Kb) and GI50 for several human cancer cell was observed.

Stabilized microbubbles (microspheres) are widely used to enhance the contrast of ultrasound imaging. Our data provide direct evidence that the contrast agents, Levovist, PVC-AN (polyvinylidene chloride-acrylonitryl copolymer), and Albunex (compared to 5% human albumin), at concentrations comparable to those used for ultrasound imaging, enhance H2O2 production (through the superoxide-dependent pathway) in air-saturated aqueous solutions exposed to 47 kHz ultrasound above the cavitation threshold. These agents also act as scavengers of .H atoms and .OH radicals, thus lowering H2O2 formation (by recombination of .OH radicals) in argon-saturated solutions. EPR spin trapping also reveals that secondary radicals derived from the contrast agents are produced by reactions with .H and .OH which are formed by pyrolysis of water inside cavitation bubbles. In addition, the contrast agents themselves undergo pyrolysis reactions in the cavitation bubbles as demonstrated by formation of methyl radicals. Possible deleterious consequences of the formation of sonochemical intermediates may have to be assessed, particularly since some of the echo contrast agents have been shown to lower the cavitation threshold of diagnostic ultrasound. Unlike the microspheres formed from organic molecules, inorganic microspheres, Eccospheres, because of their stability and inert nature with respect to participation in free radical processes, appear to be suitable tools for enhancing the yields of aqueous sonochemical reactions.

Although free radicals have been reported to play a role in the expansion of ischemic brain lesions, the effect of free radical scavengers is still under debate. In this study, the temporal profile of ischemic stroke lesion sizes was assessed for more than one year to evaluate the effect of edaravone which might reduce ischemic damage.

MPTP is a neurotoxin thought to damage dopaminergic neurons through free radical formation. MPTP is metabolized in the brain to MPP(+), which is taken up into dopaminergic neurons via the dopamine transporter and assumed to impair mitochondrial function. We used striatal synaptosomes and telencephalic mitochondria to further investigate MPP(+) mechanism of action. For comparison, the respiratory toxins FCCP, a cyanide analog that uncouples mitochondrial ATP production, and rotenone, a NADH dehydrogenase inhibitor, were also tested. FCCP, MPP(+) and rotenone caused a rapid but stable decrease in [3H]dopamine (DA) uptake by striatal synaptosomes. Two free radical scavengers, the salen-manganese complex EUK-134, and the spin trap s-PBN, did not prevent MPP(+)-induced decrease in DA uptake. However, addition of ATP during synaptosome preparation resulted in partial recovery of MPP(+)-induced [3H]DA uptake decrease. Generation of oxygen free radicals by treatment of telencephalic mitochondria with MPP(+), FCCP, or rotenone, was evaluated by measuring DCF fluorescence, while light emission by the luciferin-luciferase complex was used to determine ATP levels. MPP(+), unlike rotenone, did not produce oxygen free radicals, but rather blocked ATP production in mitochondria, as did FCCP and rotenone. Taken together, these results suggest that MPP(+) toxicity, at least during its initial stages, is primarily due to a decrease in ATP synthesis by mitochondria and not to free radical formation.

Acetaminophen overdoses cause cell injury in the liver. It is widely accepted that liver toxicity is initiated by the reactive N-acetyl-para-aminophenol (APAP) metabolite N-acetyl-p-benzoquinone imine (NAPQI), which first depletes glutathione and then irreversibly binds to mitochondrial proteins and nuclear DNA. As a consequence, mitochondrial respiration is inhibited, and DNA strands break. NAPQI also promotes the oxidative stress since glutathione is one of the main free-radical scavengers in the cell. However, so far it is unknown where exactly free radicals are generated. In this study, we used relaxometry, a novel technique that allows nanoscale magnetic resonance imaging detection of free radicals. The method is based on fluorescent nanodiamonds, which change their optical properties based on their magnetic surrounding. To achieve subcellular resolution, these nanodiamonds were targeted to cellular locations, that is, the cytoplasm, mitochondria, and the nucleus. Since relaxometry is sensitive to spin noise from radicals, we were able to measure the radical load in these different organelles. For the first time, we measured APAP-induced free-radical production in an organelle-specific manner, which helps predict and better understand cellular toxicity.

Cancer is a complex interaction among multiple signaling pathways involving a variety of target molecules. Cancer causes morbidity and mortality in millions of people worldwide, and due to its prevalence, the discovery of novel anticancer drugs is urgently required. Nature is considered an important source of the discovery of anticancer treatments, and many of the cytotoxic medicines in clinics today are derived from plants and other natural sources. Reactive oxygen species (ROS) induce a variety of human cancers, and antioxidants or scavengers are used to counteract them. The current study reports on the screening of extracts from 57 plants that are used in the galilee district as a food and/or for traditional medicine. Investigating the free radical scavenging capacity and these plants, and their cytotoxicity, may prove helpful to high-throughput screening projects that use antioxidants and cytotoxic natural products. The current study assessed the correlation between free radical scavenging and cytotoxicity. Correlational analysis is important for increasing the efficiency of the screening process. In the present study, free radical scavenging was assessed using a DPPH assay, while cytotoxicity was measured using a XTT assay. A total of 9 extracts were indicated to exhibit EC50 values <250 µg/ml, and 4 others exhibited a high antioxidant content, with EC50 values, for free radical scavenging, of <0.5 µg/ml. An in-depth analysis of the results revealed that the extracts of plants that exhibit an EC50 of free radical scavenging ≤10 µg/ml show a degree of enrichment toward increased cytotoxicity. It is recommended that future studies test the validity of the conclusions of the current study on other cancer cell-lines, and isolate and identify the bioactive agents that are found in the most cytotoxic extracts of plants.