Thioredoxin, a singlet oxygen quencher and hydroxyl radical scavenger: redox independent functions

Thioredoxin is a ubiquitous small protein known to protect cells and tissues against oxidative stress. However, its exact antioxidant nature has not been elucidated. In this report, we present evidence that human thioredoxin is a powerful singlet oxygen quencher and hydroxyl radical scavenger. Human thioredoxin at 3 microM caused 50% inhibition of TEMP-(1)O(2) (TEMPO) adduct formation in a photolysis EPR study. In contrast, Escherichia coli thioredoxin caused 50% inhibition of TEMPO formation at 80 microM. Both E. coli thioredoxin and human thioredoxin inhibited (*)OH dependent DMPO-OH formation as demonstrated by EPR spectrometry. The quenching of (1)O(2) or scavenging of (*)OH was not dependent upon the redox state of thioredoxin. Using a human thioredoxin in which the structural cysteines were mutated to alanine, Trx-C3A, we show that structural cysteines that do not take part in the catalytic functions of the protein are also important for its reactive oxygen scavenging properties. In addition, using a quadruple mutant Trx-C4A, where one of the catalytic cysteines, C35 was mutated to alanine in addition to the mutated structural cysteines, we demonstrated that catalytic cysteines are also required for the scavenging action of thioredoxin. Identification of thioredoxin as a (1)O(2) quencher and (*)OH scavenger may be of significant importance in explaining various redox-related antioxidant functions of thioredoxin.     

Vasoactive intestinal peptide, a singlet oxygen quencher

The neuropeptide vasoactive intestinal peptide (VIP), a highly basic 28-amino acid peptide, has a widespread distribution in the body. The functional specificity of this peptide not only includes its potent vasodilatory activity, but also its role in protecting lungs against acute injury, in preventing T-lymphocyte proliferation and in modulating immune function. We have investigated the possible antioxidant properties of VIP and found that VIP does not have significant O2-, OH., or H2O2 scavenging ability. However, VIP was found to inhibit, in a dose-dependent manner, the 1O2-dependent 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) formation. 1O2 was produced in photosensitizing systems using rose bengal or methylene blue as sensitizers and was detected as TEMP-1O2 product (TEMPO) by electron paramagnetic resonance (EPR) spectroscopic techniques. The formation of TEMPO signal was strongly inhibited by known singlet quenchers, e.g. beta-carotene, histidine as well as azide, but not by catalase (20 micrograms/ml) which removes H2O2 and mannitol (6 mM) or ethanol (5.9 mM) which remove OH.. Superoxide dismutase (2.5 micrograms/ml) inhibited the photoreaction up to 20% by removing O2- and most probably by blocking the secondary charge transfer pathway of 1O2 formation. These results suggest that the formation of nitroxide radical by 1O2 attack on TEMP may be used as a simple and specific assay for 1O2, and VIP can serve as an effective 1O2 scavenger/quencher, thus it may modulate the oxidative tissue injury caused by this reactive species of oxygen.     

Manganese complexes of curcumin analogues: evaluation of hydroxyl radical scavenging ability, superoxide dismutase activity and stability towards hydrolysis

In order to improve the antioxidant property of curcumin and its analogue, diacetylcurcumin, manganese was incorporated into the structures in order to enhance superoxide dismutase (SOD) activity. Manganese (Mn) complexes of curcumin (CpCpx) and diacetylcurcumin (AcylCpCpx) were synthesized and firstly investigated for SOD activity and hydroxyl radical (HO*) scavenging ability. SOD activity was evaluated by both the nitroblue tetrazolium (NBT) reduction assay and electron paramagnetic resonance (EPR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as a spin trapping agent. CpCpx and AcylCpCpx inhibited the NBT reduction and decreased the DMPO/OOH adduct much greater than corresponding antioxidants or ligands, with IC50 values of 29.9 and 24.7 microM (NBT), and 1.09 and 2.40 mM (EPR), respectively. For EPR, potassium superoxide (KO2) was used as a source of O2- where qualitative results suggested that CpCpx and AcylCpCpx were SOD mimics, which catalyze the conversion of O2- to dioxygen and hydrogen peroxide (H2O2). Additionally, CpCpx and AcylCpCpx exhibited the great inhibition of DMPO/OH adduct formation with an IC50 of 0.57 and 0.37mM, respectively, which were comparable to that of curcumin (IC50 of 0.64 mM), indicating that both Mn complexes are also an effective HO* scavenger. The stability against hydrolysis in water, various buffers and human blood/serum was carried out in vitro. It was found that both Mn complexes were pH and salt concentration dependent, being more stable in basic pH. In the human blood/serum test, CpCpx was more stable against hydrolysis than AcylCpCpx with about 10 and 20% of free Mn2+ releasing, respectively.     

Hydroxyl radical scavenging and singlet oxygen quenching properties of polyamines

Polyamines (cadaverine, putrescine, spermidine, spermine) have been shown to be present in all prokaryotic and eukaryotic cells, and proposed to be important anti-inflammatory agents. Some polyamines at high concentrations are known to scavenge superoxide radicals in vitro. We have investigated the possible antioxidant properties of polyamines and found that polyamines, e.g., cadaverine, putrescine, spermidine and spermine do not scavenge superoxide radicals at 0.5, 1.0 and 2 mM concentrations. However, polyamines were found to be potent scavengers of hydroxyl radicals. Hydroxyl radicals were produced in a Fenton type reaction and detected as DMPO-OH adducts by electron paramagnetic resonance spectroscopic technique. Spermine, spermidine, putrescine and cadaverine inhibited DMPO-OH adduct formation in a dose dependent manner, and at 1.5 mM concentration virtually eliminated the adduct formation. The *OH-dependent TBA reactive product of deoxyribose was also inhibited by polyamines in a dose-dependent manner. Polyamines were also found to inhibit the 1O2-dependent 2,2,6,6-tetramethylpiperidine N-oxy 1 (TEMPO) formation. 1O2 was produced in a photosensitizing system using Rose Bengal or Methylene Blue as photosensitizers, and was detected as TEMP-1O2 adduct by EPR spectroscopy. Spermine or spermidine inhibited the 1O2-dependent TEMPO formation maximally to 50%, whereas putrescine or cadaverine inhibited this reaction only up to 15%, when used at 0.5 and 1 mM concentrations. These results suggest that polyamines are powerful. OH scavengers, and spermine or spermidine also can quench singlet oxygen at higher concentrations.     

On-line EPR study of free radicals induced by peroxidase/H(2)O(2) in human low-density lipoprotein

The aim of this study was to use direct electron paramagnetic resonance (EPR) spectroscopy at 37 degrees C and spin trapping techniques to study radical species formed during horseradish peroxidase/H(2)O(2)-initiated low-density lipoprotein (LDL) oxidation. Using direct EPR, we obtained evidence for the formation not only of the alpha-tocopheroxyl radical but also of a protein radical(s), assigned to a tyrosyl radical(s) of apolipoprotein B-100 (apo B-100). Spin trapping with 2-methyl-2-nitrosopropane revealed (i) the formation of a mobile adduct with beta-hydrogen coupling assigned to a lipid radical and (ii) a partially immobilised adduct detected in LDL as well as in apo B-100, assigned after proteolytic digestion to the trapping of a radical centred on a tertiary carbon atom of an aromatic residue, probably tyrosine. Our results support the hypothesis that radicals are initiators of the oxidative process, and show that their formation is an early event in peroxidase-mediated oxidation. We also tested the effects of resveratrol (RSV), a polyphenolic antioxidant present in red wine. Our data indicate that 1-10 microM RSV is able to accelerate alpha-tocopherol consumption, conjugated dienes formation and the decay kinetics of LDL-centred radicals. Since phenols are substrates for peroxidases, this result may be ascribed to a RSV-mediated catalysis of peroxidase activity.     

Curcumin-glutathione interactions and the role of human glutathione S-transferase P1-1

Curcumin (diferuloylmethane), a yellow pigment of turmeric with antioxidant properties has been shown to be a cancer preventative in animal studies. It contains two electrophilic alpha, beta-unsaturated carbonyl groups, which can react with nucleophilic compounds such as glutathione (GSH), but formation of the GSH-curcumin conjugates has not previously been demonstrated. In the present studies, we investigated the reactions of curcumin with GSH and the effect of recombinant human glutathione S-transferase(GST)P1-1 on reaction kinetics. Glutathionylated products of curcumin identified by FAB-MS and MALDI-MS included mono- and di-glutathionyl-adducts of curcumin as well as cyclic rearrangement products of GSH adducts of feruloylmethylketone (FMK) and feruloylaldehyde (FAL). The presence of GSTP1-1 significantly accelerated the initial rate of GSH-mediated consumption of curcumin in 10 mM potassium phosphate, pH 7.0, and 1 mM GSH. GSTP1-1 kinetics determined using HPLC indicated substrate inhibition (apparent K(m) for curcumin of 25+/-11 microM, and apparent K(i) for curcumin of 8+/-3 microM). GSTP1-1 was also shown to catalyze the reverse reaction leading to the formation of curcumin from GSH adducts of FMK and FAL.     

EPR spin trapping evaluation of ROS production in human fibroblasts exposed to cerium oxide nanoparticles: Evidence for NADPH oxidase and mitochondrial stimulation

To better understand the antioxidant (enzyme mimetic, free radical scavenger) versus oxidant and cytotoxic properties of the industrially used cerium oxide nanoparticles (nano-CeO(2)), we investigated their effects on reactive oxygen species formation and changes in the antioxidant pool of human dermal and murine 3T3 fibroblasts at doses relevant to chronic inhalation or contact with skin. Electron paramagnetic resonance (EPR) spin trapping with the nitrone DEPMPO showed that pretreatment of the cells with the nanoparticles dose-dependently triggered the release in the culture medium of superoxide dismutase- and catalase-inhibitable DEPMPO/hydroxyl radical adducts (DEPMPO-OH) and ascorbyl radical, a marker of ascorbate depletion. This DEPMPO-OH formation occurred 2 to 24h following removal of the particles from the medium and paralleled with an increase of cell lipid peroxidation. These effects of internalized nano-CeO(2) on spin adduct formation were then investigated at the cellular level by using specific NADPH oxidase inhibitors, transfection techniques and a mitochondria-targeted antioxidant. When micromolar doses of nano-CeO(2) were used, weak DEPMPO-OH levels but no loss of cell viability were observed, suggesting that cell signaling mechanisms through protein synthesis and membrane NADPH oxidase activation occurred. Incubation of the cells with higher millimolar doses provoked a 25-60-fold higher DEPMPO-OH formation together with a decrease in cell viability, early apoptosis induction and antioxidant depletion. These cytotoxic effects could be due to activation of both the mitochondrial source and Nox2 and Nox4 dependent NADPH oxidase complex. Regarding possible mechanisms of nano-CeO(2)-induced free radical formation in cells, in vitro EPR and spectrophotometric studies suggest that, contrary to Fe(2+) ions, the Ce(3+) redox state at the surface of the particles is probably not an efficient catalyst of hydroxyl radical formation by a Fenton-like reaction in vivo.     

Indomethacin inactivates gastric peroxidase to induce reactive-oxygen-mediated gastric mucosal injury and curcumin protects it by preventing peroxidase inactivation and scavenging reactive oxygen

We have investigated the mechanism of indomethacin-induced gastric ulcer caused by reactive oxygen species (ROS) and the gastroprotective effect of curcumin thereon. Curcumin dose-dependently blocks indomethacin-induced gastric lesions, showing 82% protection at 25 mg/kg. Indomethacin-induced oxidative damage by ROS as shown by increased lipid peroxidation and thiol depletion is almost completely blocked by curcumin. Indomethacin causes nearly fivefold increase in hydroxyl radical (()OH) and significant inactivation of gastric mucosal peroxidase to elevate endogenous H(2)O(2) and H(2)O(2)-derived ()OH, which is prevented by curcumin. In vitro studies indicate that indomethacin inactivates peroxidase irreversibly only in presence of H(2)O(2) by acting as a suicidal substrate. 5,5-Dimethyl-pyrroline-N-oxide (DMPO) protects the peroxidase, indicating involvement of indomethacin radical in the inactivation. Indomethacin radical was also detected in the peroxidase-indomethacin-H(2)O(2) system as DMPO adduct (a(N) = 15 G, a(beta)(H) = 16 G) by electron spin resonance spectroscopy. Curcumin protects the peroxidase in a concentration-dependent manner and consumes H(2)O(2) for its oxidation as a suitable substrate of the peroxidase, thereby blocking indomethacin oxidation. Curcumin can also scavenge ()OH in vitro. We suggest that curcumin protects gastric damage by efficient removal of H(2)O(2) and H(2)O(2) -derived ()OH by preventing peroxidase inactivation by indomethacin.     

Concentration dependent antioxidant/pro-oxidant activity of curcumin studies from AAPH induced hemolysis of RBCs

The antioxidant properties of curcumin have been studied by evaluating its ability to protect RBCs from AAPH (2,2'-azobis (2-amidinopropane) hydrochloride) induced oxidative damage. RBCs are susceptible to oxidative damage, resulting in peroxidation of the membrane lipids, release of hemoglobin (hemolysis), release of intracellular K(+) ions and depletion of glutathione (GSH). In this paper, lipid peroxidation, hemolysis and K(+) ion loss in RBCs were assessed respectively by formation of thiobarbituric acid reactive substances (TBARS), absorbance of hemoglobin at 532nm and flame photometry. The treatment of RBCs with curcumin showed concentration dependant decrease in level of TBARS and hemolysis. The IC(50) values for inhibition of lipid peroxidation and hemolysis were estimated to be 23.2+/-2.5 and 43+/-5microM respectively. However in contrast to the above mentioned effects, curcumin in similar concentration range, did not prevent release of intracellular K(+) ions during the process of hemolysis, rather curcumin induced its release even in the absence of hemolysis. The ability of curcumin to prevent oxidation of intracellular GSH due to hemolysis showed mixed results. At low concentrations of curcumin (<10microM) it prevented GSH depletion and at higher concentrations, the GSH levels decreased gradually. Curcumin scavenges the peroxyl radical generated from AAPH. Based on these results, it is concluded that curcumin exhibits both antioxidant/pro-oxidant activity, in a concentration dependent manner.     

Curcumin inhibits formation of amyloid beta oligomers and fibrils, binds plaques, and reduces amyloid in vivo

Alzheimer's disease (AD) involves amyloid beta (Abeta) accumulation, oxidative damage, and inflammation, and risk is reduced with increased antioxidant and anti-inflammatory consumption. The phenolic yellow curry pigment curcumin has potent anti-inflammatory and antioxidant activities and can suppress oxidative damage, inflammation, cognitive deficits, and amyloid accumulation. Since the molecular structure of curcumin suggested potential Abeta binding, we investigated whether its efficacy in AD models could be explained by effects on Abeta aggregation. Under aggregating conditions in vitro, curcumin inhibited aggregation (IC(50) = 0.8 microM) as well as disaggregated fibrillar Abeta40 (IC(50) = 1 microM), indicating favorable stoichiometry for inhibition. Curcumin was a better Abeta40 aggregation inhibitor than ibuprofen and naproxen, and prevented Abeta42 oligomer formation and toxicity between 0.1 and 1.0 microM. Under EM, curcumin decreased dose dependently Abeta fibril formation beginning with 0.125 microM. The effects of curcumin did not depend on Abeta sequence but on fibril-related conformation. AD and Tg2576 mice brain sections incubated with curcumin revealed preferential labeling of amyloid plaques. In vivo studies showed that curcumin injected peripherally into aged Tg mice crossed the blood-brain barrier and bound plaques. When fed to aged Tg2576 mice with advanced amyloid accumulation, curcumin labeled plaques and reduced amyloid levels and plaque burden. Hence, curcumin directly binds small beta-amyloid species to block aggregation and fibril formation in vitro and in vivo. These data suggest that low dose curcumin effectively disaggregates Abeta as well as prevents fibril and oligomer formation, supporting the rationale for curcumin use in clinical trials preventing or treating AD.     

Probing interactions from solvent-exchangeable protons and monovalent cations with the 1,2-propanediol-1-yl radical intermediate in the reaction of dioldehydrase

The reaction of adenosylcobalamin-dependent dioldehydrase with 1,2-propanediol gives rise to a radical intermediate observable by EPR spectroscopy. This reaction requires a monovalent cation such as potassium ion. The radical signal arises from the formation of a radical pair comprised of the Co(II) of cob(II)alamin and a substrate-related radical generated upon hydrogen abstraction by the 5'-deoxyadenosyl radical. The high-field asymmetric doublet arising from the organic radical has allowed investigation of its composition and environment through the use of EPR spectroscopic techniques. To characterize the protonation state of the oxygen substituents in the radical intermediate, X-band EPR spectroscopy was performed in the presence of D(2)O and compared to the spectrum in H(2)O. Results indicate that the unpaired electron of the steady-state radical couples to a proton on the C(1) hydroxyl group. Other spectroscopic experiments were performed, using either potassium or thallous ion as the activating monovalent cation, in an attempt to exploit the magnetic nature of the (205,203)Tl nucleus to identify any intimate interaction of the radical intermediate with the activating cation. The radical intermediate in complex with dioldehydrase, cob(II)alamin and one of the activating monovalent cations was observed using EPR, ENDOR, and ESEEM spectroscopy. The spectroscopic evidence did not implicate a direct coordination of the activating cation and the substrate derived radical intermediate.     

Suppression of nitric oxide oxidation to nitrite by curcumin is due to the sequestration of the reaction intermediate nitrogen dioxide, not nitric oxide

Curcumin, a phytochemical with antioxidant and other cytoprotective properties, has been reported to reduce nitrite formation during nitric oxide (NO) oxidation in solution. This decrease in nitrite production was attributed to the direct sequestration of NO by curcumin. In this report, we confirm that curcumin inhibits nitrite formation from DEA/NO-derived NO in a concentration-dependent manner. However, curcumin over a concentration range of 3-50 microM had no effect on the concentration of free NO (0.5 microM) in solution at 37 degrees C as assessed using an NO electrode. We conclude that the inhibitory effect of curcumin on the oxidation of NO to nitrite is due to its known sequestration of the reaction intermediate nitrogen dioxide (NO(2)). The ability of curcumin to sequester NO(2), but not NO, suggests that curcumin may be useful for separating the actions of NO(2) from those of NO in various biological systems.     

Curcumin treatment enhances islet recovery by induction of heat shock response proteins, Hsp70 and heme oxygenase-1, during cryopreservation

Limited recovery of islets post-cryopreservation influences graft survival and transplantation efficiency during diabetes treatment. As curcumin, a potent antioxidant/radical scavenging compound, protects islets against beta cell toxins, we hypothesized that inclusion of curcumin during cryopreservation or during post-thaw culture or both may rescue islets from cryoinjury. To test the effect of curcumin inclusion on islet recovery murine islets were isolated by the collagenase digestion, cultured for 48 h, cryopreserved using dimethylsulphoxide as cryoprotectant -- with or without curcumin (10 microM) -- and then slow cooled to -40 degrees C before immersing them in liquid nitrogen for 7 days. Following rapid thawing with sucrose gradient and 24 h post-thaw culture -- in presence or absence of curcumin (10 microM) -- islet viability and functionality were determined. Islet recovery in curcumin treated groups was significantly higher than in groups where islets were cryopreserved without curcumin. Islets cryopreserved with curcumin also showed more intact islets as well as better morphology as compared to islets cryopreserved without curcumin. Curcumin treated islets also showed significant inhibition of ROS generation as compared to islets cryopreserved without curcumin. Glucose responsiveness and insulin secretion in islets cryopreserved with curcumin was equal to that of the freshly isolated islets as against islets cryopreserved without curcumin. Elevated level of Hsp 70 and HO-1 were observed in islets cryopreserved with curcumin and may contribute to curcumin-induced islet rescue. Hence, we conclude that inclusion of curcumin into cryopreservation medium inhibits ROS generation and corresponding islet damage and dysfunction.     

EPR kinetic studies of superoxide radicals generated during the autoxidation of 1-methyl-4-phenyl-2,3-dihydropyridinium, a bioactivated intermediate of parkinsonian-inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

1-Methyl-4-phenyl-2,3-dihydropyridinium (MPDP+), a metabolic product of the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), has been shown to generate superoxide radicals during its autoxidation process. The generation of superoxide radicals was detected as a 5,5-dimethyl-1-pyrroline-N-oxide (DMPO).O2- spin adduct by spin trapping in combination with EPR techniques. The rate of formation of spin adduct was dependent not only on the concentrations of MPDP+ and oxygen but also on the pH of the system. Superoxide dismutase inhibited the spin adduct formation in a dose-dependent manner. The ability of DMPO to trap superoxide radicals, generated during the autoxidation of MPDP+, and of superoxide dismutase to effectively compete with this reaction for the available O2-, has been used as a convenient competition reaction to quantitatively determine various kinetic parameters. Thus, using this technique the rate constant for scavenging of superoxide radical by superoxide dismutase was found to be 7.56 x 10(9) M-1 s-1. The maximum rate of superoxide generation at a fixed spin trap concentration using different amounts of MPDP+ was found to be 4.48 x 10(-10) M s-1. The rate constant (K1) for MPDP+ making superoxide radical was found to be 3.97 x 10(-6) s-1. The secondary order rate constant (KDMPO) for DMPO-trapping superoxide radicals was found to be 10.2 M-1 s-1. The lifetime of superoxide radical at pH 10.0 was calculated to be 1.25 s. These values are in close agreement to the published values obtained using different experimental techniques. These results indicate that superoxide radicals are produced during spontaneous oxidation of MPDP+ and that EPR spin trapping can be used to determine the rate constants and lifetime of free radicals generated in aqueous solutions. It appears likely that the nigrostriatal toxicity of MPTP/MPDP+ leading to Parkinson's disease may largely be due to the reactivity of these radicals.     

Biological evaluation of curcumin and related diarylheptanoids

Nine derivatives of three natural diarylheptanoids, curcumin, demethoxycurcumin and bisdemethoxycurcumin, were prepared. Their antioxidant, free radical scavenging, nitric oxide (NO) inhibitory and cytotoxic activities were evaluated and compared with those of the respective natural compounds. Curcumin (1), demethoxycurcumin (2), demethyldemethoxy-curcumin (C3), diacetyldemethoxycurcumin (AC2) and triacetyldemethylcurcumin (AC5) exhibited higher antioxidant activity than quercetin while products from demethylation of 1 and 2 exhibited higher free radical scavenging activity. Compounds AC2 and AC5 were found to be most active in inhibiting breast cancer cells (MCF-7) proliferation with IC50 values of 6.7 and 3.6 microM, respectively. The activity of AC2 is almost doubled and of AC5 almost tripled as compared to curcumin. Their selectivity towards different cell lines is also more noticeable. Compounds AC2 and AC5 also showed increased activity against a human prostate cancer cell line (DU-145) and non-small lung cancer cell line (NCI-H460) with IC50 values of 20.4, 16.3 and 18.3, 10.7 microM, respectively.     

Antioxidant activity of galloyl quinic derivatives isolated from P. lentiscus leaves

The antioxidant properties of galloyl quinic derivatives isolated from Pistacia lentiscus L. leaves have been investigated by means of Electron Paramagnetic Resonance spectroscopy (EPR) and UV-Vis spectrophotometry. Antioxidant properties have been also estimated using the biologically relevant LDL test. The scavenger activities of gallic acid, 5- O -galloyl, 3,5- O -digalloyl, 3,4,5- O -trigalloyl quinic acid derivatives, have been estimated against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, superoxide ( O 2 - ) radical, and hydroxyl (OH) radical. On the whole, the scavenger activity raised as the number of galloyl groups on the quinic acid skeleton increased. The half-inhibition concentrations (IC 50 ) of di- and tri-galloyl derivatives did not exceed 30 μM for all the tested free radicals. All the tested metabolites strongly reduced the oxidation of low-density lipoproteins (LDL), following a trend similar to that observed for the scavenger ability against OH radical.     

On-line EPR study of free radicals induced by peroxidase/H2O2 in human low-density lipoprotein

The aim of this study was to use direct electron paramagnetic resonance (EPR) spectroscopy at 37 °C and spin trapping techniques to study radical species formed during horseradish peroxidase/H₂O₂-initiated low-density lipoprotein (LDL) oxidation. Using direct EPR, we obtained evidence for the formation not only of the α-tocopheroxyl radical but also of a protein radical(s), assigned to a tyrosyl radical(s) of apolipoprotein B-100 (apo B-100). Spin trapping with 2-methyl-2-nitrosopropane revealed (i) the formation of a mobile adduct with β-hydrogen coupling assigned to a lipid radical and (ii) a partially immobilised adduct detected in LDL as well as in apo B-100, assigned after proteolytic digestion to the trapping of a radical centred on a tertiary carbon atom of an aromatic residue, probably tyrosine. Our results support the hypothesis that radicals are initiators of the oxidative process, and show that their formation is an early event in peroxidase-mediated oxidation. We also tested the effects of resveratrol (RSV), a polyphenolic antioxidant present in red wine. Our data indicate that 1–10 μM RSV is able to accelerate α-tocopherol consumption, conjugated dienes formation and the decay kinetics of LDL-centred radicals. Since phenols are substrates for peroxidases, this result may be ascribed to a RSV-mediated catalysis of peroxidase activity.     

Curcumin protects against ovariectomy-induced bone loss and decreases osteoclastogenesis

Curcumin has anti-oxidative activity. In view of the increasing evidence for a biochemical link between increased oxidative stress and reduced bone density we hypothesized that curcumin might increase bone density by elevating antioxidant activity in some target cell type. We measured bone density by Micro-CT, enzyme expression levels by quantitative PCR or enzyme activity, and osteoclast (OC) formation by tartrate-resistant acid phosphatase staining. The bone mineral density of the femurs of curcumin-administered mice was significantly higher than that of vehicle-treated mice after ovariectomy (OVX) and this was accompanied by reduced amounts of serum collagen-type I fragments, which are markers of bone resorption. Curcumin suppressed OC formation by increasing receptor activator of nuclear factor-κB ligand (RANKL)-induced glutathione peroxidase-1, and reversed the stimulatory effect of homocysteine, a known H(2) O(2) generator, on OC formation by restoring Gpx activity. Curcumin generated an aberrant RANKL signal characterized by reduced expression of nuclear factor of activated T cells 2 (NFAT2) and attenuated activation of mitogen-activated protein kinases (ERK, JNK, and p38). Curcumin thus inhibited OVX-induced bone loss, at least in part by reducing osteoclastogenesis as a result of increased antioxidant activity and impaired RANKL signaling. These findings suggest that bone loss associated with estrogen deficiency could be attenuated by curcumin administration.     

Antioxidant and radical scavenging properties of curcumin

Curcumin (diferuoyl methane) is a phenolic compound and a major component of Curcuma longa L. In the present paper, we determined the antioxidant activity of curcumin by employing various in vitro antioxidant assays such as 1,1-diphenyl-2-picryl-hydrazyl free radical (DPPH*) scavenging, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging activity, N,N-dimethyl-p-phenylenediamine dihydrochloride (DMPD) radical scavenging activity, total antioxidant activity determination by ferric thiocyanate, total reducing ability determination by the Fe(3+)-Fe(2+) transformation method, superoxide anion radical scavenging by the riboflavin/methionine/illuminate system, hydrogen peroxide scavenging and ferrous ions (Fe(2+)) chelating activities. Curcumin inhibited 97.3% lipid peroxidation of linoleic acid emulsion at 15 microg/mL concentration (20 mM). On the other hand, butylated hydroxyanisole (BHA, 123 mM), butylated hydroxytoluene (BHT, 102 mM), alpha-tocopherol (51 mM) and trolox (90 mM) as standard antioxidants indicated inhibition of 95.4, 99.7, 84.6 and 95.6% on peroxidation of linoleic acid emulsion at 45 microg/mL concentration, respectively. In addition, curcumin had an effective DPPH* scavenging, ABTS*(+) scavenging, DMPD*(+) scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, ferric ions (Fe(3+)) reducing power and ferrous ions (Fe(2+)) chelating activities. Also, BHA, BHT, alpha-tocopherol and trolox, were used as the reference antioxidant and radical scavenger compounds. According to the present study, curcumin can be used in the pharmacological and food industry because of these properties.     

Antioxidant activity of an aminothiazole compound: possible mechanisms

Oceans are among the richest natural sources of many bioactive compounds. Several of these compounds have shown pharmacological activities for many diseases. Dendrodoine (5-[(3-N-dimethylamino)-1,2,4-thiadiazolyl]-3-indanyl methanone) is an alkaloid extracted from the marine tunicate Dendrodoa grossularia. Aminothiazoles have a wide range of biological activities including anti-tumor and antioxidant properties. The aim of our study was to examine the antioxidant ability of an aminothiazole derivative, dendrodoine analogue (DA) [(4-amino-5-benzoyl-2-(4-methoxy phenylamino) thiazole] which has been chemically synthesized and is similar to dendrodoine. In all the biochemical assays used in our study, corresponding to different levels of protection, DA showed concentration dependent antioxidant ability. DA (3.07 microM) showed an ability to inhibit 2,2'-azobis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) radical formation to the extent of 0.17 microM of 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). The ferric complex reducing ability of 3.07 microM DA was equivalent to 110 microM Trolox. 3.07 microM DA gave 84% protection against deoxyribose degradation, a measure of hydroxyl radical scavenging. DA also has an ability to scavenge NO radical, 3.07 microM DA effecting 20% scavenging. Concentration dependent inhibition of lipid peroxidation and protein oxidation induced by 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and ascorbate-Fe2+ was observed with low concentrations of DA (1.5-3.07 microM). Mechanistic studies using pulse radiolysis revealed that DA scavenges peroxyl radicals with a bimolecular rate constant of 3 x 10(8)M(-1)s(-1). Moreover, the initially formed nitrogen-centered radical gets transformed into sulfur-centered radical before furnishing any final product. Our results indicated that DA can be a free radical scavenger and potential antioxidant for future application.