Reactive Oxygen Species Scavenging Effects Research Articles

Inhibitory effects of melatonin on the lipopolysaccharide‐induced CC chemokine expression in BV2 murine microglial cells are mediated by suppression of Akt‐induced NF‐κB and STAT/GAS activity

Melatonin influences sleep and circadian rhythm, and it has anti-inflammatory functions. However, the mechanism of its anti-inflammatory roles is not well understood. In our studies, we show that melatonin blocked lipopolysaccharide (LPS)-induced CCL2 (monocyte chemotactic protein-1; MCP-1), CCL5 (Regulated upon Activation, Normal T-cell Expressed, and Secreted), and CCL9 (macrophage inflammatory protein-1γ) chemokine mRNA expression in BV2 murine microglial cells. Melatonin markedly inhibited LPS-induced Akt phosphorylation and NF-κB activation. Furthermore, melatonin inhibited LPS-induced STAT1/3 phosphorylation and interferon-gamma activated sequence (GAS)-driven transcriptional activity. Interestingly, these effects were not associated with reactive oxygen species scavenging effects of melatonin or melatonin receptor signal pathways. Taken together, our results suggested that melatonin has anti-inflammatory functions through down-regulation of chemokine expression by inhibition of NF-κB and STAT/GAS activation in LPS-stimulated BV2 murine microglial cell line.

Curcumin Alters Membrane Phase-Separation, Particle Size Distribution, Permeability, and Anisotropy Differently in Unsaturated or Saturated Small Unilamellar Vesicles

Curcumin is an extract of turmeric, and is an effective reactive oxygen species scavenger, with potential anti-cancer benefits. In this study, we examined the effects of curcumin on membrane bilayer permeability, fusion, anisotropy, hydrodynamic radius and negative staining EM using two lipid vesicle delivery systems: 1) 99% Soy phosphatidylcholine (Soy PC) and 2) DMPC/DPPG/Cholesterol (1:1:0.7 mol/mol/mol) (MPC). Curcumin was added to these two vesicle populations at 10:1, 50:1, and 100:1 (lipid mol/curc mol). The structure of curcumin suggests it would most likely intercalate into the head group region of the membrane bilayer, and thus our hypothesis was that the unsaturated fatty acids of Soy PC vesicles would accommodate curcumin with less membrane perturbation than the saturated MPC vesicles. Curcumin increased membrane permeability to carboxyfluorescein leakage (CF) in both Soy PC and MPC small unilamellar vesicles in a concentration-dependent manner; however, Soy PC vesicles were 35% more permeable to CF than MPC vesicles. The fusion of the Soy PC and MPC was examined by vesicle mixing using the Tb/DPA-assay. Both Soy PC and MPC vesicles fused within seconds of addition, and the rate of fusion was dependent on buffer calcium concentration. Curcumin had membrane concentration-dependent effects on the fluorescence anisotropy of DPH-PC and TMA-DPH Soy PC and MPC vesicles. Lipid vesicle hydrodynamic radius and TEM negative stained vesicles indicate a unimodal distribution for Soy PC vesicles (r = 100-120 nm), while MPC vesicles had a bimodal distribution (r = 100-130 nm and 3000-4000 nm). Thus curcumin can more readily intercalate into more unsaturated lipid vesicle bilayers, and we conclude that for maximal delivery of curcumin to cells using a liposomal therapy, a more unsaturated vesicle system is suggested.

Peroxyacetyl nitrate-induced oxidative and calcium signaling events leading to cell death in ozone-sensitive tobacco cell-line

It has long been concerned that some secondary air pollutants such as smog components, ozone (O3) and peroxyacetyl nitrate (PAN), are highly phytotoxic even at low concentrations. Compared with the biology of O3, we largely lack the information on the toxicity model for PAN at the cellular signaling levels. Here, we studied the cell-damaging impact of PAN using suspension culture of smog-sensitive tobacco variety (Bel-W3). The cells were exposed to freshly synthesized PAN and the induced cell death was assessed under microscope after staining with Evans blue. Involvement of reactive oxygen species (ROS) in PAN toxicity was suggested by PAN-dependently increased intracellular H2O2 and also by the cell-protective effects of ROS scavengers and related inhibitors. Calcium chelator also lowered the level of PAN-induced cell death, indicating that Ca2+ is also involved. Using a transgenic cell line expressing aequorin, an increase in cytosolic Ca2+ concentration responsive to the pulse of PAN, but sensitive to Ca2+ channel blockers, was recorded, indicating that Ca2+ channels are activated by PAN or PAN-derived signals. Above data show some similarity between the signaling mechanisms responsive to O3 and PAN.

Nebivolol

Nebivolol is a highly selective β1-adrenoceptor blocker, which also stimulates endothelial nitric oxide synthase and scavenges reactive oxygen species (ROS). These characteristics endow nebivolol, compared with conventional β-blockers, with a favorable hemodynamic profile for the treatment of hypertension, chronic heart failure, and possibly other cardiovascular diseases. Nebivolol is a racemic mixture of d- and l-isomers. Its β1-antagonistic properties reside primarily with d-nebivolol although both isomers are capable of increasing the release of NO from the endothelium after binding to β2- or β3-adrenergic receptors. The latter action results in vasodilatation and reduced vascular resistance. Nebivolol also scavenges ROS in a receptor-independent manner by direct interaction with free radicals. By scavenging ROS nebivolol not only reduces oxidant stress but also augments NO bioavailability. The endothelial nitric oxide synthase -stimulating and ROS scavenging effects of nebivolol act synergistically to provide cardiovascular protection in addition to its β1-antagonistic action.

Fomitoside-K from <i>Fomitopsis nigra</i> Induces Apoptosis of Human Oral Squamous Cell Carcinomas (YD-10B) <i>via</i> Mitochondrial Signaling Pathway

In this study, a new lanostane triterpene glycoside (fomitoside-K) having biologically active molecules was isolated from a mushroom Fomitopsis nigra to test its anticancer activity on human oral squamous cell carcinomas (YD-10B). We focused on the effect of fomitoside-K on apoptosis, the mitochondria-mediated death pathway and the accumulation of reactive oxygen species (ROS) in YD-10B cells. Fomitoside-K could induce a dose and time-dependent apoptosis in YD-10B cells as characterized by cell morphology, cell cycle arrest, inhibition of survivin, activation of poly(ADP-ribose) polymerase (PARP), caspase-3, -9 and an increased expression ratio of Bax/Bcl-2. The mitochondria membrane potential loss and cytochrome c (Cyt C) release from mitochondria to cytosol were observed during the induction. Moreover, fomitoside-K caused dose-dependent elevation of intracellular ROS level and increase phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in YD-10B cells. To further investigate the mechanisms, we examined the effects of ROS scavenger N-acetyl-L-cysteine (NAC) and selective inhibitors for mitogen activated protein kinase (MAPK) pathways on the cell death. The fomitoside-K induced cell death by ROS was significantly inhibited by NAC, ERK (PD98059) and JNK inhibitor (SP600125). In addition, fomitoside-K has a synergistic effect with adriamycin in suppressing the growth of YD-10B cells. These data suggest that fomitoside-K induces apoptosis in YD-10B cells through the ROS-dependent mitochondrial dysfunction pathway and provides a mechanistic framework for further exploring the use of fomitoside-K against the proliferation of human oral cancer.

The neuroprotective effects of electrolyzed reduced water and its model water containing molecular hydrogen and Pt nanoparticles

Human brain is the biggest energy consuming tissue in human body. Although it only represents 2% of the body weight, it receives 20% of total body oxygen consumption and 25% of total body glucose utilization. For that reason, brain is considered to be the most vulnerable part of human body against the reactive oxygen species (ROS), a by-product of aerobic respiration. Oxidative stress is directly related to a series of brain dysfunctional disease such as Alzheimer’ sd isease, Parkinson’s disease etc. Electrolyzed reduced water (ERW) is a functional drinking water containing a lot of molecular hydrogen and a small amount of platinum nanoparticles (Pt NPs, Table 1). ERW is known to scavenge ROS and protect DNA from oxidative damage [1]. We previously showed that ERW was capable of extending lifespan of Caenorhabditis elegans by scavenging ROS [2]. Molecular hydrogen could scavenge ROS and protected brain from oxidative stress [3]. Pt NPs are also a new type of multi-functional ROS scavenger [4]. Materials and methods In this research, we used TI-200S ERW derived from 2 mM NaOH solution produced by a batch type electrolysis device and model waters containing molecular hydrogen and synthetic Pt NPs of 2-3 nm sizes as research models of ERW to examine the anti-oxidant capabilities of ERW on several kinds of neural cells such as PC12, N1E115, and serum free mouse embryo (SFME) cells. We pretreated the ERW and 200 μM H2O2 and examined the neuroprotective effects of ERW on PC12, N1E115 and SFME cells, using WST-8 method. We also examined the intracellular ROS scavenging effects of ERW on N1E115 cells after pretreated cells with ERW and H2O2 using DCFH-DA. We checked the protective effects of ERW on mitochondria and cytoplasm by Rh123 and Fuo-3 AM stain. We also examined the ATP production of SFME cells after pretreated with ERW and H2O2 by Bioluminescence Assay Kit. Finally, we used dissolved hydrogen (DH) and Pt NPs as research models to examine their neuroprotective effects. Results

Cytotoxic Effects of Curcumin At Various Concentrations and Role of Curcumin on Lipid Peroxidation and Activities of Antioxidant Enzymes of the Rat Peripheral Blood Lymphocytes

Abstract 4933 Background:Curcumin (diferuloylmethane) is an active ingredient of turmeric and has been suggested to have antiproliferative, pro-oxidant and cytotoxic effects (Das R, Apoptosis 2008). Curcumin has also been shown to have antioxidant effects (Rastogi M, Free Radic Res 2008). Our aim was to assess the cytotoxic, pro-oxidant and antioxidant effects of curcumin on rat peripheral blood lymphocytes (RPL) in culture. Methods:Different concentrations of curcumin (0.325 μM, 0.65 μM, 1.3 μM) were analyzed for cytotoxic effects on rat peripheral lymphocytes. The cytotoxic effect of curcumin was assessed by measuring cell viability using Trypan blue exclusion assay. DNA damage by curcumin (at 0.65 μM) was confirmed by the COMET assay. The effect of curcumin (at 0.65 μM) on oxidation was assessed by measuring the activity of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and lipid peroxidation (LP) by spectrophotometry. RPLs were cultured for 72 hr in vitro. The pro-oxidant effect of curcumin was determined by comparing LP induction in control (PBS treated) vs curcumin treated RPLs. Parameters were measured at 0, 24 and 48 hrs of curcumin exposure. Reactive oxygen species (ROS) generated by treatment with FeCl3 (10 mM) and H2O2 (5 mM) (Fe/H) were used to demonstrate the ROS scavenging effect of curcumin. Lymphocyte cultures were treated with either Fe/H alone or in combination with Curcumin. Oxidation levels were assessed as described above at 0, 24, 48 hrs following exposure. Results:Cytotoxic effects were seen at different curcumin concentrations (Table 1). Viability of RPLs were 42% at 0.64 μM concentration vs 72% at 0.325 μM. 30% decrease in cell viability of RPLs were seen at 0.65 μM concentration compared to 0.325 μM. Hence, 0.65 μM concentration was used for further experiments. There was a significant increase in DNA damage in curcumin exposed lymphocytes at 48hr (table 2). There was a significant increase in LP in curcumin exposed RPLs compared to control lymphocytes. By contrast, there was no significant change in LP values in presence of Fe/H at all time points compared to control. There was a significant decrease in LP when RPL cultures were incubated with curcumin for 48 hr compared to 0 hr of incubation. There was significant increase in activities of SOD, catalase and GPx in presence of curcumin and Fe/H (table 3 and 4).Table 1:Cytotoxic effect of curcumin as demonstrated Trypan blue viability assay Cnt=controlLymphocyte cultureLymphocyte cell viability at 48 hr (%)Cnt84Curcumin 0.325 μM72Curcumin 0.65 μM42Curcumin 1.3 μM35Table 2:Cytotoxic effect of curcumin (at 0.65 μM) as demonstrated by % Tail DNA. Two sample t-test was used to calculate p value% tail DNA (mean ± SD)Cnt (at 48 hr) (n= 6)0.53 ± 0.71Curcumin exposed lymphocyte (at 48 hr) (n=4)32.96 ± 17.97Difference in % tail DNA between control and Curcumin at 48 hrsp = 0.0019Table 3:Activities of antioxidant enzymes- SOD, catalase, GPx and LP level in optical density (OD- mean of 3 set of cultures)Lymphocyte culture (C) ODOD at 0hrOD at 24hrOD at 48hrProoxidant Activity (n=3)Only curcuminLP0.03100.11300.08500.05067Antioxidant activity (n=3)PBS+ FeCl3+H2O2Curcumin. + FeCl3+H2O2LP0.02200.02500.01600.01266SOD0.05600.02300.05400.13333Catalase0.02000.09330.22300.0936GPx0.01200.03630.12330.2170Table 4:Comparisons for enzyme activities between cnt and curcumin. One way ANOVA test was used to derive p values.NS= no significantComparison of enzyme activities between Cnt and Curcumin (at 0, 24, 48 hr)Comparison of enzymes activities (in presence of curcumin and Fe/H) at various time pointsCnt- 0hrcnt-24hrcnt-48hr0-24 hr0-48 hrPro-oxidant effectLP<0.0001<0.0001<0.0002NSNSAntioxidant effectCulture+ Fe/H (control) Vs Curcumin. + Fe/HComparison of enzymes activities (in presence of Curcumin and Fe/H) at various time pointsLPNSNSNSNS0.0392SODNSNS<0.00280.0015<0.0006Catalase<0.0053<0.0001<0.00510.0009NSGPx<0.0249<0.0001<0.0001<0.0001<0.0001 Conclusions:Dose dependent cytotoxic and DNA damage effects of curcumin are seen in RPLs. Treatment with curcumin alone increases lipid peroxidation, However in the presence of oxidative stress, curcumin may act as an antioxidant (ROS scavenging effect) by stimulating activities of antioxidant enzyme and preventing lipid peroxidation. Disclosures:No relevant conflicts of interest to declare.

Latex of Euphorbia Antiquorum Induces Apoptosis in Human Cervical Cancer Cells via c-Jun N-terminal Kinase Activation and Reactive Oxygen Species Production

Latex of Euphorbia antiquorum (EA) has inhibitory effects on several different cancer cell lines. However, the molecular mechanism of EA inhibitory effects on human cervical cancer HeLa cell growth has not been explored. EA induced apoptosis, which was characterized by morphological change, DNA fragmentation, increased sub-G1 population, and alterations in levels of apoptosis-associated proteins. Treatment with EA increased cell death and expression levels of caspase-8, -9, and -3. EA suppressed expression of Bcl-2, increased Bax, and reduced cleavage of Bid and the translocation of tBid to the mitochondria and the release of cytochrome c from mitochondria. EA caused a loss of mitochondrial membrane potential (ΔΨm) and an increase in cellular reactive oxygen species (ROS). EA-induced ROS formation was suppressed by cyclosporine A (an inhibitor of the ΔΨm) or allopurinol (an effective scavenger of ROS). EA also increased expression of Fas, FasL, and c-Jun N-terminal kinase (JNK), p38, and mitogen-activated protein kinase (MAPK) and decreased expression of extracellular signal-regulated kinase (ERK) 1/2-p. Co-treatment with the JNK inhibitor SP600125 inhibited EA-induced apoptosis and the activation of caspase-8, -9, and -3. Results of this study provide support for the hypothesis that EA causes cell death via apoptotic pathways in human cervical adenocarcinoma HeLa cells.

Global Inhibition of Reactive Oxygen Species (ROS) Inhibits Paclitaxel-Induced Painful Peripheral Neuropathy

Paclitaxel (Taxol®) is a widely used chemotherapeutic agent that has a major dose limiting side-effect of painful peripheral neuropathy. Currently there is no effective therapy for the prevention or treatment of chemotherapy-induced painful peripheral neuropathies. Evidence for mitochondrial dysfunction during paclitaxel-induced pain was previously indicated with the presence of swollen and vacuolated neuronal mitochondria. As mitochondria are a major source of reactive oxygen species (ROS), the aim of this study was to examine whether pharmacological inhibition of ROS could reverse established paclitaxel-induced pain or prevent the development of paclitaxel-induced pain. Using a rat model of paclitaxel-induced pain (intraperitoneal 2 mg/kg paclitaxel on days 0, 2, 4 & 6), the effects of a non-specific ROS scavenger, N-tert-Butyl-α-phenylnitrone (PBN) and a superoxide selective scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) were compared. Systemic 100 mg/kg PBN administration markedly inhibited established paclitaxel-induced mechanical hypersensitivity to von Frey 8 g and 15 g stimulation and cold hypersensitivity to plantar acetone application. Daily systemic administration of 50 mg/kg PBN (days −1 to 13) completely prevented mechanical hypersensitivity to von Frey 4 g and 8 g stimulation and significantly attenuated mechanical hypersensitivity to von Frey 15 g. Systemic 100 mg/kg TEMPOL had no effect on established paclitaxel-induced mechanical or cold hypersensitivity. High dose (250 mg/kg) systemic TEMPOL significantly inhibited mechanical hypersensitivity to von Frey 8 g & 15 g, but to a lesser extent than PBN. Daily systemic administration of 100 mg/kg TEMPOL (day −1 to 12) did not affect the development of paclitaxel-induced mechanical hypersensitivity. These data suggest that ROS play a causal role in the development and maintenance of paclitaxel-induced pain, but such effects cannot be attributed to superoxide radicals alone.

The ROS scavenging and renal protective effects of pH-responsive nitroxide radical-containing nanoparticles

The ultimate objective of nanoparticle-based therapy is to functionalize nanomedicines in a micro-disease environment without any side effects. Here, we reveal that our pH-responsive nitroxide radical-containing nanoparticles (RNP pH) disintegrate within the renal acidic lesion and act as scavengers of reactive oxygen species (ROS), leading to a relief of acute kidney injury (AKI). RNP pH was prepared using amphiphilic block copolymers possessing 2,2,6,6-tetramethylpiperidine- N-oxyl (TEMPO) moieties via amine linkage as a side chain of the hydrophobic segment. The self-assembled RNP pH disintegrated at pH below 7.0 because of a protonation of the amino groups in the hydrophobic core of the nanoparticles, thereby resulting in an improvement in ROS scavenging activity. Using a renal ischemia-reperfusion AKI model in mice, the therapeutic effect of RNP pH on ROS damage was evaluated. Unlike the RNP without pH-triggered disintegration (RNP Non−pH), the RNP pH showed extremely high ROS scavenging activity and renal protective effects. It is interesting to note that the side effect of nitroxide radicals was markedly suppressed due to the compartmentalization of nitroxide radicals in the core of RNP pH in untargeted area. The morphology changes in RNP pH were confirmed by analyzing electron spin resonance spectra, and these findings provide the evidence of the real therapeutic effect of the environment-sensitive specific disintegration of nanoparticles in vivo.

No protective effect of curcumin on hydrogen peroxide-induced cytotoxicity in HepG2 cells

Scavenging of intracellular reactive oxygen species (ROS) is one of the potential mechanisms contributing to the protective effects of many antioxidants. Curcumin, a natural product, is an effective ROS scavenger. However, the role of its ROS scavenging ability in its cytoprotective action remains to be clarified. Herein, the protective effects of curcumin on hydrogen peroxide (H2O2)- and tert-butyl hydroperoxide-induced ROS formation and HepG2 cell injury were determined. HepG2 cells were pretreated with curcumin for 30min and then treated with H2O2 (500µM) or tert-butyl hydroperoxide (200µM) for 24h. Curcumin pretreatment dramatically decreased H2O2- and tert-butyl hydroperoxide-induced ROS production, but failed to suppress cytotoxicity of those compounds. H2O2 induced decreases in mitochondrial membrane potential (ΔΨm) and increases in DNA fragmentation could not be reversed by curcumin. Furthermore, curcumin enhanced expression of H2O2-induced pro-apoptotic protein Bax expression and inhibited expression of anti-apoptotic proteins Bcl-2 and Bcl-xL. In addition, curcumin significantly decreased p38MAPK and phospho-CDC-2 protein expression and increased phospho-p38MAPK, p42/44MAPK, and phospho-p42/44MAPK protein expression. These results suggest that short pretreatment and subsequent longer co-treatment of low concentrations of curcumin showed no obvious protective effect on H2O2-induced HepG2 cell injury.

Spontaneous ultraweak photon emission imaging of oxidative metabolic processes in human skin: effect of molecular oxygen and antioxidant defense system

All living organisms emit spontaneous ultraweak photon emission as a result of cellular metabolic processes. In this study, the involvement of reactive oxygen species (ROS) formed as the byproduct of oxidative metabolic processes in spontaneous ultraweak photon emission was studied in human hand skin. The effect of molecular oxygen and ROS scavengers on spontaneous ultraweak photon emission from human skin was monitored using a highly sensitive photomultiplier tube and charged coupled device camera. When spontaneous ultraweak photon emission was measured under anaerobic conditions, the photon emission was decreased, whereas under hyperaerobic condition the enhancement in photon emission was observed. Spontaneous ultraweak photon emission measured after topical application of glutathione, α-tocopherol, ascorbate, and coenzyme Q10 was observed to be decreased. These results reveal that ROS formed during the cellular metabolic processes in the epidermal cells play a significant role in the spontaneous ultraweak photon emission. It is proposed that spontaneous ultraweak photon emission can be used as a noninvasive tool for the temporal and spatial monitoring of the oxidative metabolic processes and intrinsic antioxidant system in human skin.

Role of Oxidant Scavengers in the Prevention of Ca2+ Homeostasis Disorders

A number of disorders, such as Alzheimer disease and diabetes mellitus, have in common the alteration of the redox balance, resulting in an increase in reactive oxygen species (ROS) generation that might lead to the development of apoptosis and cell death. It has long been known that ROS can significantly alter Ca2+ mobilization, an intracellular signal that is involved in the regulation of a wide variety of cellular functions. Cells have a limited capability to counteract the effects of oxidative stress, but evidence has been provided supporting the beneficial effects of exogenous ROS scavengers. Here, we review the effects of oxidative stress on intracellular Ca2+ homeostasis and the role of antioxidants in the prevention and treatment of disorders associated to abnormal Ca2+ mobilization induced by ROS.

Effect of N-Acetyl Cysteine and α-Linolenic Acid on Sulfur Mustard Caused Impairment of In Vitro Endothelial Tube Formation

Sulfur mustard (SM), an alkylating chemical warfare agent, leads to tissue damage, including inflammation, blister formation, and impaired wound healing. Especially wound healing is of concern because after SM exposure, wound healing is prolonged. In this study, we focused on the effect of SM (30 and 100μM) on endothelial tube formation, apoptosis, and proliferation in mouse embryoid bodies (EBs), which provide an appropriate model for investigating vasculogenesis and angiogenesis. EBs were exposed to SM for 30 min on day 0, 3, or 6 of EBs' growth, were allowed to grow until day 7, then fixed, and immunostained (PECAM-1, Ki67, and activated caspase-3). SM significantly decreased endothelial tube formation compared with unexposed EBs. Additionally, we observed a significant increase of apoptosis. As the formation of reactive oxygen species (ROS) is discussed to be involved in the pathophysiology of SM toxicity, we evaluated the effect of ROS scavengers (α-linolenic acid [ALA] and N-acetyl cysteine [NAC]) in the same experimental setup. Temporary effects of both scavengers could be detected, in particular NAC seemed to have temporary significant positive effects on endothelial tube formation in 100μM SM-exposed EBs. ALA augmented proliferation when administered after 30μM SM exposure on day 3, whereas NAC treatment on day 0 decreased apoptosis induced by 100μM SM. Taken together, our findings pointed to a negative effect of SM on vascularization and endothelial tube formation. ROS scavengers NAC and ALA showed temporary, but not long-lasting, rescuing effects regarding endothelial tube formation after SM exposure.

Antioxidant activity of unexplored indole derivatives: Synthesis and screening

The present study envisaged the development of novel antioxidant candidates using the indole scaffold. Several tryptophan and tryptamine derivatives were synthesized, in particular prenylated indole compounds, and their scavenging activity for reactive oxygen species (ROS) and reactive nitrogen species (RNS) was investigated. The library substitution pattern included several alkyl chains at positions N-1, C-2 of the indole nucleus, including prenyl and isopentyl chain, as well as different groups at the side chain (C-3) that allowed the investigation of a possible radical stabilization. The results obtained showed that tryptophan ( 8), tryptamine ( 9), N-phthaloyl tryptamine ( 5) and N-prenyl tryptophan ( 13) were the most active against peroxyl radical (ROO •) with activities higher than Trolox, which was used as control. The scavenging of hypochlorous acid (HOCl) was also evaluated and tryptophan ( 8) and tryptamine ( 9) showed IC 50 of 3.50 ± 0.4 and 6.00 ± 0.60 μM, respectively. Significant activity was also found for the N-prenyl tryptophan ( 13) with an IC 50 of 4.13 ± 0.17 μM and C-2 prenylated derivative ( 14), with 4.56 ± 0.48 μM. The studies were extended to RNS and best results were obtained against peroxynitrite anion (ONOO − ) in the presence of NaHCO 3. N-alkylated tryptophan ( 18) showed a high activity with an IC 50 of 14.0 ± 6.8 μM. The results show that the tested compounds are effective scavengers of ROS and RNS, and suggest that the radical stabilization is strongly dependent on the type of substituents on the indolic moiety and on their relative positions. In addition, the radical dissipation inside the indolic system is mandatory for the observed antioxidant activity.

Spermine pretreatment confers dehydration tolerance of citrus in vitro plants via modulation of antioxidative capacity and stomatal response

Polyamines, small aliphatic polycations, have been suggested to play key roles in a number of biological processes. In this paper, attempts were made to investigate the possibility of improving dehydration tolerance of citrus in vitro plants by exogenous application of spermine (Spm). 'Red Tangerine' (Citrus reticulata Blanco) in vitro plants pretreated with 1 mM Spm exhibited less wilted phenotype and lower water loss and electrolyte leakage than the control under dehydration. Spm-pretreated plants contained higher endogenous polyamine content during the course of the experiment relative to the control, particularly at the end of dehydration, coupled with higher expression levels of ADC and SPMS. Histochemical staining showed that the Spm-pretreated leaves were stained to a lower extent than those without Spm pretreatment, implying generation of less reactive oxygen species (ROS). On the contrary, activities of peroxidase (POD) and superoxide dismutase (SOD) in the Spm-pretreated samples were higher than the control at a given time point or during the whole experiment, suggesting that Spm exerted a positive effect on antioxidant systems. In addition, significantly smaller stomatal aperture size was observed in Spm-pretreated epidermal peels, which showed that stomatal closure was promoted by polyamines. All of these data suggest that Spm pretreatment causes accumulation of higher endogenous polyamines and accordingly leads to more effective ROS scavenging (less tissue damage) and stimulated stomatal closure (lower water loss) upon dehydration, which may function collectively to enhance dehydration tolerance.

Scirpusin A, a hydroxystilbene dimer from Xinjiang wine grape, acts as an effective singlet oxygen quencher and DNA damage protector

Grapes and red wines are rich sources of phenolic compounds such as anthocyanins, catechins, flavonols and stilbenes, most of which are potent antioxidants showing cardioprotective properties. We first isolated scirpusin A, a hydroxystilbene dimer, from a wine grape of Xinjiang, and studied its antioxidant activity. Reactive oxygen species scavenging effects and the protection against reactive singlet oxygen-induced DNA damage of scirpusin A have been investigated in our experiments. The concentration of scirpusin A required to inhibit 50% of (1)O(2) generation was 17 micromol L(-1), while addition of scirpusin A at 140 micromol L(-1) caused complete inhibition. Further kinetic study revealed that the reaction of Scirpusin A with singlet oxygen has an extremely high rate constant (k(a) = 4.68 x 10(9) L mol(-1) s(-1)). Scirpusin A (140 micromol L(-1)) exhibited significant inhibition effects on pBR322 DNA breakage. However, scavenging effects of scirpusin A on superoxide anion O(2) (*-) and hydroxyl radical .OH were not potent as the inhibitor rates at a concentration of 1400 micromol L(-1) were 28.83% and 19.5%, respectively. The present study shows that scirpusin A is a selective quencher of singlet oxygen and a protector against reactive singlet oxygen-induced pBR322 DNA damage at very low concentrations.

Effect of ketorolac and diclofenac on the impairment of endothelium-dependent relaxation induced by reactive oxygen species in rabbit abdominal aorta

BackgroundReactive oxygen species (ROS) induce lipid peroxidation and tissue damage in endothelium. We studied the influences of ketorolac and diclofenac on ROS effects using the endothelium of rabbit abdominal aorta.MethodsIsolated rabbit aortic rings were suspended in an organ bath filled with Krebs-Henseleit (K-H) solution bubbled with 5% CO2 and 95% O2 at 37.5℃. After being stimulated to contract with phenylephrine (PE, 10-6 M), changes in arterial tension were recorded following the cumulative administration of acetylcholine (ACh, 3 × 10-8 to 10-6 M). The percentages of ACh-induced relaxation of aortic rings before and after exposure to ROS, generated by electrolysis of K-H solution, were used as the control and experimental values, respectively. The aortic rings were pretreated with ketorolac or diclofenac at the same concentrations (10-5 M to 3 × 10-4 M), and the effects of these agents were compared with the effects of ROS scavengers: catalase, mannitol, sodium salicylate and deferoxamine and the catalase inhibitor, 3-amino-1,2,4-triazole (3AT).ResultsBoth ketorolac and diclofenac maintained endothlium-dependent relaxation induced by ACh in a dose-related manner inspite of ROS attack (P < 0.05 vs. control value). The 3AT pretreated ketorolac (3 × 10-3 M) group was decreased more significantly than un-pretreated ketorolac (P < 0.05).ConclusionsThese findings suggest that ketorlac and diclofenac preserve the endothelium-dependent vasorelaxation against the attack of ROS, in a concentration-related manner. One of the endothelial protection mechanisms of ketorolac may be hydrogen peroxide scavenging.

Anti-ischemic and anti-inflammatory activity of (S)-cis-verbenol

(S)-cis-verbenol, a natural metabolite from (-)-alpha-pinene of host pine tree, has been suggested to have anti-ischemic activity. However, the exact mechanism for the anti-ischemic activity of (S)-cis-verbenol remains unclear yet. In the present study, (S)-cis-verbenol reduced cerebral ischemic injury caused by 1.5-h middle cerebral artery occlusion followed by 24-h reperfusion. Furthermore, (S)-cis-verbenol significantly prevented neuronal cell death caused by oxygen-glucose deprivation (OGD, 1 h) and subsequent re-oxygenation (5 h). While (S)-cis-verbenol did not inhibit the NMDA-stimulated calcium influx, it reduced the intracellular level of reactive oxygen species (ROS) elevated by OGD/re-oxygenation. ORAC assay indicated that (S)-cis-verbenol potently eliminated peroxyl radicals. In DPPH and DHR123 fluorescence assays, however, (S)-cis-verbenol did not show a direct ROS scavenging effect. Furthermore, (S)-cis-verbenol reduced the expression levels of pro-inflammatory cytokines in ischemic brain and immunostimulated glial cells. The present results indicate that (S)-cis-verbenol may be a useful therapeutic agent due to its anti-oxidative and anti-inflammatory activities.

Oryzadine, a new alkaloid of Oryza sativa cv. Heugjinjubyeo, attenuates oxidative stress-induced cell damage via a radical scavenging effect

The antioxidant properties of oryzadine, a new alkaloid, obtained from Oryza sativa cv. Heugjinjubyeo was investigated by applying various methods based on cell-free and cell experiments. Oryzadine showed scavenging effects on the hydroxyl radical, superoxide radical, and intracellular reactive oxygen species (ROS). Oryzadine inhibited H2O2-induced DNA damage, which was demonstrated by DNA tail formation, lipid peroxidation which was demonstrated by the formation of thiobarbituric acid reactive substance (TBARS), and protein oxidation which was demonstrated by protein carbonyl formation. Therefore, oryzadine protected H2O2-induced cell damage. Our results show that the cytoprotective effects of oryzadine stem from its ability to inhibit H2O2-induced apoptosis, as demonstrated by a decrease in apoptotic body formation and the inhibition of mitochondrial membrane potential (ΔΨ) loss. Taken together, these findings suggest that oryzadine protected cells against H2O2-induced cell damage via ROS scavenging effect. Therefore, oryzadine could be considered a significant natural source of antioxidant.