Identification of chemical structure and free radical scavenging activity of diphlorethohydroxycarmalol isolated from a brown alga, Ishige okamurae

To obtain a natural antioxidant from a marine biomass, this study investigated the antioxidative activity of methanolic extracts from the marine brown alga, Ishige okamurae collected off Jeju Island. A potent free radical scavenging activity was detected in the ethyl acetate fraction containing polyphenolic compounds, and the potent antioxidant elucidated as a kind of phlorotannin, diphlorethohydroxycarmalol, by NMR and mass spectroscopic data. The free radical scavenging activities of the diphlorethohydroxycarmalol were investigated in relation to 1,1-diphenyl-2-picrylhydrazyl (DPPH), alkyl, and hydroxyl radicals using an electron spin resonance (ESR) system. The diphlorethohydroxycarmalol was found to scavenge DPPH (IC50=3.41 microM) and alkyl (IC50=4.92 microM) radicals more effectively than the commercial antioxidant, ascorbic acid. Therefore, these results present diphlorethohydroxycarmalol as a new phlorotannin with a potent antioxidative activity that could be useful in cosmetics, foods, and pharmaceuticals.     

Phenylpropanoid amides from Alisma orientalis and their protective effects against H2O2-induced damage in SH-SY5Y cells

Five new phenylpropanoid amides, including N-trans-feruloyl-N′-cis-feruloyl-cadaverine (1), N,N′-trans-diferuloyl-3-oxo-cadaverine (2), N-trans-feruloyl-N′-cis-feruloyl-3-hydroxy-cadaverine (3), N,N′-cis-diferuloyl-3-hydroxy-cadaverine (4), N-trans-p-coumaroyl-N′-trans-feruloyl-3-hydroxy-cadaverine (5), were isolated from Alisma orientalis together with four known analogues. Their structural elucidations were conducted by using 1D and 2D NMR and HRESIMS spectroscopic analyses. The isolated compounds were assayed for their inhibitory activities against HCE-2, anti-oxidant effects, and their protective effects on H₂O₂-induced damage in human dopaminergic neuroblastoma cells (SH-SY5Y). Compounds 3, 6, and 7 displayed moderate anti-oxidant activities with IC₅₀ values in the range of 36.9–40.7 μM. Compound 5 showed significant protective activity, while compounds 1, 2, 4, 7, and 8 showed moderate protective activities.     

Seven new triterpenoids from the aerial parts of Ilex cornuta and protective effects against H2O2-induced myocardial cell injury

Seven new triterpenoids (1–7), together with two known ones (8–9), were isolated from the aerial parts ofIlex cornuta. The leaves of I. cornuta are the major source of “Kudingcha”, a popular herbal tea consumed in China and other countries. The structures of compounds 1–7 were determined as 20-epi-urs-12,18-dien-28-oic acid 3β-O-α-l-arabinopyranoside (1), 20-epi-urs-12,18-dien-28-oic acid 2′-O-acetyl-3β-O-α-l-arabinopyranoside (2), 20-epi-urs-12,18-dien-28-oic acid 3β-O-β-d-glucuronopyranoside-6-O-methyl ester (3), 3β,23-dihydroxy-20-epi-urs-12,18-dien-28-oic acid (4), 23-hydroxy-20-epi-urs-12,18-dien-28-oic acid 3β-O-α-l-arabinopyranoside (5), 23-hydroxy-20-epi-urs-12,18-dien-28-oic acid 3β-O-β-d-glucuronic acid (6), 23-hydroxy-20-epi-urs-12,18-dien-28-oic acid 3β-O-β-d-glucuronopyranoside-6-O-methyl ester (7), on the basis of spectroscopic analyses (IR, ESI–MS, HR-ESI–MS, 1D and 2D NMR) and chemical reactions. Protective effects against H₂O₂-induced H9c2 cardiomyocyte injury were tested in vitro for compounds 1–9, and the data showed that compound 4 had significant cell-protective effect. Compounds 1-9 did not show significant DPPH radical scavenging activity.     

Crosstalk between JNK and SUMO signaling pathways: deSUMOylation is protective against H2O2-induced cell injury

Background

Oxidative stress is a key feature in the pathogenesis of several neurological disorders. Following oxidative stress stimuli a wide range of pathways are activated and contribute to cellular death. The mechanism that couples c-Jun N-terminal kinase (JNK) signaling, a key pathway in stress conditions, to the small ubiquitin-related modifier (SUMO), an emerging protein in the field, is largely unknown.

Methodology/Principal Findings

With this study we investigated if SUMOylation participates in the regulation of JNK activation as well as cellular death in a model of H₂O₂ induced-oxidative stress. Our data show that H₂O₂ modulates JNK activation and induces cellular death in neuroblastoma SH-SY5Y cells. Inhibition of JNK''s action with the D-JNKI1 peptide rescued cells from death. Following H₂O₂, SUMO-1 over-expression increased phosphorylation of JNK and exacerbated cell death, although only in conditions of mild oxidative stress. Furthermore inhibition of SUMOylation, following transfection with SENP1, interfered with JNK activation and rescued cells from H₂O₂ induced death. Importantly, in our model, direct interaction between these proteins can occur.

Conclusions/Significance

Taken together our results show that SUMOylation may significantly contribute to modulation of JNK activation and contribute to cell death in oxidative stress conditions.     

Antioxidant potential of aminothiazole derivative and its protective effect on H2O2-induced oxidative damage on pBR322 DNA and RBC cellular membrane

The present work was carried out to evaluate the antioxidant and free radical scavenging activity of aminothiazole derivative by performing various in vitro assays; to study its protective effect on H(2)O(2)-induced oxidative damage on pBR322 DNA and on RBC cellular membrane. The in vitro assays were performed with different concentrations of aminothiazole derivative (6.15, 12.29, 18.44, 24.59, and 30.73 microM) and the results were compared with standards like ascorbic acid and trolox. Our results clearly indicated that aminothiazole derivative at a dose of 18.44 microM exhibited radical scavenging activity greater than that of ascorbic acid and trolox. The DNA protective effect on pBR322 DNA showed that there was a concentration-dependent inhibition of the disappearance of supercoiled (ccc) form of DNA on incubation with 30 mM H(2)O(2) in the presence of different concentrations of aminothiazole derivative. Thus our compound at 1.5 mM prevents the conversion from supercoiled (ccc) form to open circular form (oc) form of pBR322 DNA. Pretreatment with aminothiazole derivative at a dose of 18.44 microM prevents membrane damage and exhibits an IC(50) value, which is the concentration of the sample required to inhibit 50% of the radical formed greater than that of the standards (ascorbic acid and trolox). Thus our compound of interest aminothiazole derivative exhibits antioxidant and free radical scavenging properties greater than that of standards like ascorbic acid and trolox and thereby protects pBR322 DNA and RBC cellular membrane from free radical induced oxidative damage.     

Nitroxides protect horseradish peroxidase from H2O2-induced inactivation and modulate its catalase-like activity

BackgroundHorseradish peroxidase (HRP) catalyzes H₂O₂ dismutation while undergoing heme inactivation. The mechanism underlying this process has not been fully elucidated. The effects of nitroxides, which protect metmyoglobin and methemoglobin against H₂O₂-induced inactivation, have been investigated.MethodsHRP reaction with H₂O₂ was studied by following H₂O₂ depletion, O₂ evolution and heme spectral changes. Nitroxide concentration was followed by EPR spectroscopy, and its reactions with the oxidized heme species were studied using stopped-flow.ResultsNitroxide protects HRP against H₂O₂-induced inactivation. The rate of H₂O₂ dismutation in the presence of nitroxide obeys zero-order kinetics and increases as [nitroxide] increases. Nitroxide acts catalytically since its oxidized form is readily reduced to the nitroxide mainly by H₂O₂. The nitroxide efficacy follows the order 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO) > 4-OH-TPO > 3-carbamoyl proxyl > 4-oxo-TPO, which correlates with the order of the rate constants of nitroxide reactions with compounds I, II, and III.ConclusionsNitroxide catalytically protects HRP against inactivation induced by H₂O₂ while modulating its catalase-like activity. The protective role of nitroxide at μM concentrations is attributed to its efficient oxidation by P940, which is the precursor of the inactivated form P670. Modeling the dismutation kinetics in the presence of nitroxide adequately fits the experimental data. In the absence of nitroxide the simulation fits the observed kinetics only if it does not include the formation of a Michaelis-Menten complex.General SignificanceNitroxides catalytically protect heme proteins against inactivation induced by H₂O₂ revealing an additional role played by nitroxide antioxidants in vivo.     

Superoxide dismutases enhance H2O2-induced DNA damage and alter its site specificity

Superoxide dismutases (SODs) are involved in the protection of cells from oxygen toxicity. However, several papers have reported that the overexpression of CuZn-SOD causes oxidative damage to cells. We investigated a mechanism by which an excess of SODs accelerates oxidative stress. The presence of CuZn-SOD, Mn-SOD or Mn(II) enhanced the frequency of DNA damage induced by hydrogen peroxide (H2O2) and Cu(II), and altered the site specificity of the latter: H2O2 induced Cu(II)-dependent DNA damage with high frequency at the 5'-guanine of poly G sequences; when SODs were added, the frequency of cleavages at thymine and cytosine residues increased. SODs also enhanced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by H2O2 and Cu(II). We conclude that SODs may increase carcinogenic risks, e.g. of tumors in Down syndrome.     

Protective effect of salidroside against H2O2-induced cell apoptosis in primary culture of rat hippocampal neurons

Salidroside, a phenylpropanoid glycoside separated from a medicinal plant Rhodiola rosea, has been documented to have protective effects on neuronal cells in vitro. This study investigated whether salidroside was able to extend its unique neuroprotection to primary cultured rat hippocampal neurons against hydrogen peroxide (H₂O₂)-induced cell damage. Cell viability tests and cell apoptosis assays confirmed that salidroside pretreatment attenuated H₂O₂-stimulated apoptotic cell death in primary culture of hippocampal neurons in a concentration-dependent manner. The measurements of caspase-3 activity, nitric oxide (NO) production, and NO synthase (NOS) activity suggest that the protection of salidroside, shown in this study, might be mediated by inhibiting caspase-3 activity, and antagonizing NO production and NOS activity during H₂O₂ stimulation. Perhaps, this study might contribute to the development of salidroside as a broad-spectrum agent for preventing and/or treating neuronal damage in neurodegenerative disorders.     

Glutamine acts as a neuroprotectant against DNA damage, beta-amyloid and H2O2-induced stress

Glutamine is the most abundant free amino acid in the human blood stream and is 'conditionally essential' to cells. Its intracellular levels are regulated both by the uptake of extracellular glutamine via specific transport systems and by its intracellular synthesis by glutamine synthetase (GS). Adding to the regulatory complexity, when extracellular glutamine is reduced GS protein levels rise. Unfortunately, this excess GS can be maladaptive. GS overexpression is neurotoxic especially if the cells are in a low-glutamine medium. Similarly, in low glutamine, the levels of multiple stress response proteins are reduced rendering cells hypersensitive to H(2)O(2), zinc salts and DNA damage. These altered responses may have particular relevance to neurodegenerative diseases of aging. GS activity and glutamine levels are lower in the Alzheimer's disease (AD) brain, and a fraction of AD hippocampal neurons have dramatically increased GS levels compared with control subjects. We validated the importance of these observations by showing that raising glutamine levels in the medium protects cultured neuronal cells against the amyloid peptide, Aβ. Further, a 10-day course of dietary glutamine supplementation reduced inflammation-induced neuronal cell cycle activation, tau phosphorylation and ATM-activation in two different mouse models of familial AD while raising the levels of two synaptic proteins, VAMP2 and synaptophysin. Together, our observations suggest that healthy neuronal cells require both intracellular and extracellular glutamine, and that the neuroprotective effects of glutamine supplementation may prove beneficial in the treatment of AD.     

Protection by tropolones against H2O2-induced DNA damage and apoptosis in cultured Jurkat cells

Tropolones, the naturally occurring compounds responsible for the durability of heartwood of several cupressaceous trees, have been shown to possess both metal chelating and antioxidant properties. However, little is known about the ability of tropolone and its derivatives to protect cultured cells from oxidative stress-mediated damage. In this study, the effect of tropolones on hydrogen peroxide-induced DNA damage and apoptosis was investigated in cultured Jurkat cells. Tropolone, added to the cells 15 min before the addition of glucose oxidase, provided a dose dependent protection against hydrogen peroxide induced DNA damage. The IC₅₀ value observed was about 15 μM for tropolone. Similar dose dependent protection was also observed with three other tropolone derivatives such as trimethylcolchicinic acid, purpurogallin and β-thujaplicin (the IC₅₀ values were 34, 70 and 74 μM, respectively), but not with colchicine and tetramethyl purpurogallin ester. Hydrogen peroxide-induced apoptosis was also inhibited by tropolone. However, in the absence of exogenous H₂O₂ but in the presence of non-toxic concentrations of exogenous iron (100 μM Fe³⁺), tropolone dramatically increased the formation of single strand breaks at molar ratios of tropolone to iron lower than 3 to 1, while, when the ratio increased over 3, no toxicity was observed. In conclusion, the results presented in this study indicate that the protection offered by tropolone against hydrogen peroxide-induced DNA damage and apoptosis was due to formation of a redox-inactive iron complex, while its enhancement of iron-mediated DNA damage at ratios of [tropolone]/[Fe³⁺] lower than 3, was due to formation of a lipophilic iron complex which facilitates iron transport through cell membrane in a redox-active form.     

H2O2-induced changes in mitochondrial activity in isolated mouse pancreatic acinar cells

This study employed confocal laser scanning microscopy to monitor the effect of H2O2 on cytosolic as well as mitochondrial calcium (Ca2+) concentrations, mitochondrial inner membrane potential (psi m) and flavine adenine dinucleotide (FAD) oxidation state in isolated mouse pancreatic acinar cells. The results show that incubation of pancreatic acinar cells with H2O2, in the absence of extracellular Ca2+ ([Ca2+],) led to an increase either in cytosolic and in mitochondrial Ca2+ concentration. Additionally, H2O2 induced a depolarization of mitochondria and increased oxidized FAD level. Pretreatment of cells with the mitochondrial inhibitors rotenone or cyanide inhibited the response induced by H2O2 on mitochondrial inner membrane potential but failed to block oxidation of FAD in the presence of H2O2. However, the H2O2-evoked effect on FAD state was blocked by pretreatment of cells with the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP). On the other hand, perfusion of cells with thapsigargin (Tps), an inhibitor of the SERCA pump, led to an increase in mitochondrial Ca2+ concentration and in oxidized FAD level, and depolarized mitochondria. Pretreatment of cells with thapsigargin inhibited H2O2-evoked changes in mitochondrial Ca2+ concentration but not those in membrane potential and FAD state. The present results have indicated that H2O2 can evoke marked changes in mitochondrial activity that might be due to the oxidant nature of H2O2. This in turn could represent the mechanism of action of ROS to induce cellular damage leading to cell dysfunction and generation of pathologies in the pancreas.     

Natural products inspired synthesis of neuroprotective agents against H2O2-induced cell death

Stroke is a debilitating disease and the third leading cause of death in the USA, where over 2000 new stroke cases are diagnosed every day. Treatment options for stroke-related brain damage are very limited and there is an urgent need for effective neuroprotective agents to treat these conditions. Comparison of the structures of several classes of neuroprotective natural products such as limonoids and cardiac glycosides revealed the presence of a common structural motif which may account for their observed neuroprotective activity. Several natural product mimics that incorporate this shared structural motif were synthesized and were found to possess significant neuroprotective activity. These compounds enhanced cell viability against H₂O₂ induced oxidative stress or cell death in PC12 neuronal cells. The compounds were also found to enhance and modulate Na⁺/K⁺-ATPase activity of PC12 cells, which may suggest that the observed neuroprotective activity is mediated, at least partly, through interaction with Na⁺/K⁺-ATPase.     

Poly(ADP-ribose) glycohydrolase silencing protects against H2O2-induced cell death

PAR [poly(ADP-ribose)] is a structural and regulatory component of multiprotein complexes in eukaryotic cells. PAR catabolism is accelerated under genotoxic stress conditions and this is largely attributable to the activity of a PARG (PAR glycohydrolase). To overcome the early embryonic lethality of parg-knockout mice and gain more insights into the biological functions of PARG, we used an RNA interference approach. We found that as little as 10% of PARG protein is sufficient to ensure basic cellular functions: PARG-silenced murine and human cells proliferated normally through several subculturing rounds and they were able to repair DNA damage induced by sublethal doses of H2O2. However, cell survival following treatment with higher concentrations of H2O2 (0.05-1 mM) was increased. In fact, PARG-silenced cells were more resistant than their wild-type counterparts to oxidant-induced apoptosis while exhibiting delayed PAR degradation and transient accumulation of ADP-ribose polymers longer than 15-mers at early stages of drug treatment. No difference was observed in response to the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine, suggesting a specific involvement of PARG in the cellular response to oxidative DNA damage.     

Salvia macilenta exhibits antiglycating activity and protects PC12 cells against H2O2-induced apoptosis

Salvia macilenta is a member of the genus Salvia (Laminaceae) whose antioxidant activity and neuroprotective effect has been shown previously. The present study aimed to examine the antiglycating and antiapoptotic abilities of methanolic extract of this plant. Moreover, the effect of S. macilenta on neurite outgrowth and complexity after exposure to H₂O₂ has been studied. Base on our results, S. macilenta has antiglycating activity and protects PC12 cells against oxidative stress-induced apoptotic cell death, as examined by Hoechst staining and Western blot analysis of caspase-3, Bax, Bcl-2 and PARP. We further showed that S. macilenta decreased neurite growth and complexity impairment in differentiated PC12 cells exposed to oxidative stress. It caused a decrease in cell body area, neurite width, and the proportion of bipolar cells, while significantly increasing neurite length, the number of primary neurites per cell and the ratio of nodes to primary neuritis. All around, the mentioned results open a new horizon for future works to use this plant as a potential neuroprotective agent.     

Phenolic metabolites from Pyruscalleryana and evaluation of its free radical scavenging activity

Investigation of the aqueous alcoholic extract of Pyruscalleryana Decne. leaves led to the isolation of two new phenolic acids glycosides, namely protocatechuoylcalleryanin-3-O-β-glucopyranoside (1) and 3′-hydroxybenzyl-4-hydroxybenzoate-4′-O-β-glucopyranoside (2), together with nine known compounds among them lanceoloside A and methylgallate, which have been isolated for the first time from the genus Pyrus. Structures of the isolated compounds were established by spectroscopic analysis, including UV, IR, HRESI-MS, and 1D/2D NMR. The total extract and some isolated compounds were determined against DPPH (2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazinyl radical, for their free radical scavenging activity, the total alcoholic extract showed strong antioxidant activity while the two new compounds showed weak antioxidant activity.     

Cellular sites of H2O2-induced damage and their protection by nitroxides

While the exact mechanism of H2O2-induced cytotoxicity is unknown, there is considerable evidence implicating DNA as a primary target. A recent study showed that a cell-impermeable nitroxide protected mammalian cells from H2O2-induced cell killing and suggested that the protection was mediated through cell membrane-bound or extracellular factors. To further define the protective properties of nitroxides, Chinese hamster V79 cells were exposed to H2O2 with or without cell-permeable and impermeable nitroxides and selected metal chelators. EPR spectroscopy and paramagnetic line broadening agents were used to distinguish between intra- and extracellular nitroxide distribution. To study the effectiveness of nitroxide protection, in the absence of a cell membrane, H2O2-mediated damage to supercoiled plasmid DNA was evaluated. Both deferrioxamine and Tempol cross the cell membrane, and inhibited H2O2-mediated cell killing, whereas the cell-impermeable DTPA and nitroxide, CAT-1, failed to protect. Similar protective effects of the chelators and nitroxides were observed when L-histidine, which enhances intracellular injury, was added to H2O2. In contrast, when damage to plasmid DNA was induced (in the absence of a cell membrane), both nitroxides were protective. Collectively, these results do not support a role for membrane-bound or extracellular factors in mediating H2O2 cytotoxicity in mammalian cells.     

Protective effects of quercetin and its metabolites on H2O2-induced chromosomal damage to WIL2-NS cells

We investigated chromosomal damage caused by a typical flavonoid, quercetin, and its two conjugates, quercetin-3-O-sulfate and isorhamnetin, and their protective effects against chromosomal damage induced by H2O2. The chromosomal damage was detected by the cytokinesis-block micronucleus (CBMN) assay using a lymphoblastoid cell line, WIL2-NS. We found that quercetin itself induced chromosomal damage at 10 microM, but quercetin-3-O-sulfate and isorhamnetin did not induce damage up to 30 microM. In the medium used for the CBMN assay, quercetin (at 100 microM) generated a high concentration of H2O2, but the two conjugates did not at the same concentration. On the other hand, pretreatment with quercetin (at 1 microM), quercetin-3-O-sulfate (at 10 microM), and isorhamnetin (at 5 microM) prevented H2O2-induced chromosomal damage to WIL2-NS cells. These findings suggest that the induction and prevention of H2O2-induced chromosomal damage are different between quercetin and its metabolites.     

Metabolic stability and inhibitory effect of O-methylated theaflavins on H2O2-induced oxidative damage in human HepG2 cells

Seven new O-methylated theaflavins (TFs) were synthesized by using O-methyltransferase from an edible mushroom. Using TFs and O-methylated TFs, metabolic stability in pooled human liver S9 fractions and inhibitory effect on H₂O₂-induced oxidative damage in human HepG2 cells were investigated. In O-methylation of theaflavin 3′-O-gallate (TF3′G), metabolic stability was potentiated by an increase in the number of introduced methyl groups. O-methylation of TF3,3′G did not affect metabolic stability, which was likely because of a remaining 3-O-galloyl group. The inhibitory effect on oxidative damage was assessed by measuring the viability of H₂O₂-damaged HepG2 cells treated with TFs and O-methylated TFs. TF3,3′G and O-methylated TFs increased cell viabilities significantly compared with DMSO, which was the compound vehicle (p?<?0.05), and improved to approximately 100%. Only TF3′G did not significantly increase cell viability. It was suggested that the inhibitory effect on H₂O₂-induced oxidative damage was potentiated by O-methylation or O-galloylation of TFs.     

Protective effect of Nelumbo nucifera (Gaertn.) against H2O2-induced oxidative stress on H9c2 cardiomyocytes

Molecular Biology Reports - Ischemic heart disease (IHD), a severe condition of myocardium facing impediment in the supply of basic needs for cellular metabolism is caused by atherosclerosis....     

Differential protective effects of O-phenanthroline and catalase on H2O2-induced DNA damage and inhibition of protein synthesis in endothelial cells.

The respective roles of H2O2 and .OH radicals was assessed from the protective effects of catalase and the iron chelator o-phenanthroline on 1) the inhibition of protein synthesis, and 2) DNA damage and the related events (activation of the DNA repairing enzyme poly(ADP)ribose polymerase with the associated depletion of NAD and ATP stores) in cultured endothelial cells exposed to the enzyme reaction hypoxanthine-xanthine oxidase (HX-XO) or pure H2O2. Catalase added in the extracellular phase completely prevented all of these oxidant-induced changes. O-phenanthroline afforded a complete protective effect against DNA strand breakage and the associated activation of the enzyme poly(ADP)ribose polymerase. By contrast, iron chelation was only partially effective in maintaining the cellular NAD and ATP contents, as well as the protein synthetic activity. In addition, the ATP depletion following oxidant injury was much more profound than NAD depletion. These results indicate that: 1) .OH radical was most likely the ultimate O2 species responsible for DNA damage and activation of poly(ADP)ribose polymerase; 2) both H2O2 and .OH radicals were involved in the other cytotoxic effects (inhibition of protein synthesis and reduction of NAD and ATP stores); and 3) NAD and ATP depletion did not result solely from activation of poly(ADP)ribose polymerase, but other mechanisms are likely to be involved. These observations are also compatible with the existence of a compartmentalized intracellular iron pool.