Antioxidant protection of human serum albumin by chitosan

Inhibition of protein oxidation by reactive oxygen species (ROS) would confer benefit to living organisms exposed to oxidative stress, because oxidized proteins are associated with many diseases and can propagate ROS-induced damage. We measured the ability of 2800Da chitosan, D-glucosamine and N-acetyl glucosamine to protect human serum albumin from oxidation by peroxyl radicals derived from 2,2'-azobis(2-amidinopropane)dihydrochloride and N-centered radicals from 1,1'-diphenyl-2-picrylhydrazyl and from 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). Comparison with the antioxidant action of vitamin C showed that, on a molar basis, chitosan was equally effective in preventing formation of carbonyl and hydroperoxide groups in human serum albumin exposed to peroxyl radicals. It was also a potent inhibitor of conformational changes in the protein, assessed by absorption spectrum and intrinsic fluorescence. D-glucosamine was much less effective and N-acetyl glucosamine was not a useful antioxidant. Protection of the albumin from peroxyl radicals was achieved by scavenging of peroxyl radical. Chitosan was also a good scavenger of N-centered radicals, with glucosamine and N-acetyl glucosamine much less effective. The results suggest that administration of low molecular weight chitosans may inhibit neutrophil activation and oxidation of serum albumin commonly observed in patients undergoing hemodialysis, resulting in reduction of oxidative stress associated with uremia.     

Chitosan gallate as a novel potential polysaccharide antioxidant: an EPR study

A novel biopolymer-based antioxidant, chitosan conjugated with gallic acid (chitosan galloylate, chitosan-GA), is proposed. Electron paramagnetic resonance (EPR) demonstrates a wide range of antioxidant activity for chitosan-GA as evidenced from its reactions with oxidizing free radicals, that is, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), horseradish peroxidase (HRP)-H₂O₂, carbon-centered alkyl radicals, and hydroxyl radicals. The EPR spectrum of the radical formed on chitosan-GA was attributed to the semiquinone radical of the gallate moiety. The stoichiometry and effective concentration (EC₅₀) of the DPPH free radical with chitosan-GA show that the radical scavenging capacity is maintained even after thermal treatment at 100 °C for an hour. Although the degree of substitution of GA on chitosan was about 15%, its antioxidant capacity, that is, the reaction with carbon-centered and hydroxyl radicals, is comparable to that of GA.     

Formation of superoxide anion during ferrous ion-induced decomposition of linoleic acid hydroperoxide under aerobic conditions

We studied the mechanism of formation of oxygen radicals during ferrous ion-induced decomposition of linoleic acid hydroperoxide using the spin trapping and chemiluminescence methods. The formation of the superoxide anion (O2*-) was verified in the present study. The hydroxyl radical is also generated through Fenton type decomposition of hydrogen peroxide produced on disproportionation of O2*-. A carbon-centered radical was detected using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) as a spin trap. Alkoxyl radical formation is essential for the conversion of linoleic acid hydroperoxide into the peroxyl radical by ferrous ion. It is likely that the alkoxyl radical [R1CH(O*)R2] is converted into the hydroxylcarbon radical [R1C*(OH)R2] in water, and that this carbon radical reacts with oxygen to give the alpha-hydroxyperoxyl radical [R1R2C(OH)OO*], which decomposes into the carbocation [R1C+(OH)R2] and O2*-.     

Antioxidant properties of chitosan from crab shells

Crab chitosan was prepared by alkaline N-deacetylation of crab chitin for 60, 90 and 120 min and its antioxidant properties studied. Chitosan exhibited showed antioxidant activities of 58.3–70.2% at 1 mg/mL and showed reducing powers of 0.32–0.44 at 10 mg/mL. At 10 mg/mL, the scavenging ability of chitosan C60 on 1,1-diphenyl-2-picrylhydrazyl radicals was 28.4% whereas those of other chitosans were 46.4–52.3%. At 0.1 mg/mL, scavenging abilities on hydroxyl radicals were 62.3–77.6% whereas at 1 mg/mL, chelating abilities on ferrous ions were 82.9–96.5%. All EC₅₀ values of antioxidant activity were below 1.5 mg/mL. With regard to antioxidant properties assayed, the effectiveness of chitosans C60, C90 and C120 correlated with their N-deacetylation times. Overall, crab chitosan was good in antioxidant activity, scavenging ability on hydroxyl radicals and chelating abilities on ferrous ions and may be used as a source of antioxidants, as a possible food supplement or ingredient in the pharmaceutical industry.     

Oxidation of ethanol by cumene hydroperoxide in the presence of cytochrome P-450 LM2 and hemoglobin

Ethanol oxidation by cumene hydroperoxide (CHP) with participation of cytochrome P-450 LM-2 (pH 7.4) and hemoglobin (pH 7.0) was studied at 37 degrees C in phosphate buffer. Both hemoproteins form complexes with CHP that are decomposed with the liberation of the RO2., RO. and HO. radicals, thus initiating the chain oxidation of ethanol. Ethanol oxidation catalyzed by cytochrome P-450 LM-2 and hemoglobin occurs only through a radical formation and is competitively inhibited by the radical scavenging agents, e.g., 1-naphthol, thiourea, mannitol and dimethylsulfoxide (DMSO). The values of effective inhibition constants were determined for all antioxidants whose activity decreases in the following order: 1-naphthol greater than thiourea greater than mannitol greater than DMSO. The non-inhibited oxidation of ethanol in "CHP-hemoproteins" systems is characterized by low ethanol conversion because of bimolecular termination of radicals and biocatalyst destruction.     

A mechanistic study of the formation of hydroxyl radicals induced by horseradish peroxidase with NADH

During the oxidation of NADH by horseradish peroxidase (HRP-Fe(3+)), superoxide (O(-)(2)) is produced, and HRP-Fe(3+) is converted to compound III. Superoxide dismutase inhibited both the generation of O(-)(2) and the formation of compound III. In contrast, catalase inhibited only the generation of O(-)(2). Under anaerobic conditions, the formation of compound III did not occur in the presence of NADH, thus indicating that compound III is produced via formation of a ternary complex consisting of HRP-Fe(3+), NADH and oxygen. The generation of hydroxyl radicals was dependent upon O(-)(2) and H(2)O(2) produced by HRP-Fe(3+)-NADH. The reaction of compound III with H(2)O(2) caused the formation of compound II without generation of hydroxyl radicals. Only HRP-Fe(3+)-NADH (but not K(+)O(-)(2) and xanthine oxidase-hypoxanthine) was able to induce the conversion of metmyoglobin to oxymyoglobin, thus suggesting the participation of a ternary complex made up of HRP-Fe(2+…)O(2)(…)NAD(.) (but not free O(-)(2) or H(2)O(2)) in the conversion of metmyoglobin to oxymyoglobin. It appears that a cyclic pathway is formed between HRP-Fe(3+), compound III and compound II in the presence of NADH under aerobic conditions, and a ternary complex plays the central roles in the generation of O(-)(2) and hydroxyl radicals.     

Hydroxyl radicals scavenging activity of N-substituted chitosan and quaternized chitosan

N-substituted chitosan and quaternized chitosan were synthesized and their antioxidant activity against hydroxyl radicals was assessed, respectively. Compared with the antioxidant activity of chitosan, the results indicated that the two kinds of chitosan derivatives had different scavenging ability on hydroxyl radicals, which should be related to the form of amido in the two kinds of chitosan derivatives.     

A chitin synthase and its regulator protein are critical for chitosan production and growth of the fungal pathogen Cryptococcus neoformans

Chitin is an essential component of the cell wall of many fungi. Chitin also can be enzymatically deacetylated to chitosan, a more flexible and soluble polymer. Cryptococcus neoformans is a fungal pathogen that causes cryptococcal meningoencephalitis, particularly in immunocompromised patients. In this work, we show that both chitin and chitosan are present in the cell wall of vegetatively growing C. neoformans yeast cells and that the levels of both rise dramatically as cells grow to higher density in liquid culture. C. neoformans has eight putative chitin synthases, and strains with any one chitin synthase deleted are viable at 30 degrees C. In addition, C. neoformans genes encode three putative regulator proteins, which are homologs of Saccharomyces cerevisiae Skt5p. None of these three is essential for viability. However, one of the chitin synthases (Chs3) and one of the regulators (Csr2) are important for growth. Cells with deletions in either CHS3 or CSR2 have several shared phenotypes, including sensitivity to growth at 37 degrees C. The similarity of their phenotypes also suggests that Csr2 specifically regulates chitin synthesis by Chs3. Lastly, both chs3Delta and the csr2Delta mutants are defective in chitosan production, predicting that Chs3-Csr2 complex with chitin deacetylases for conversion of chitin to chitosan. These data suggest that chitin synthesis could be an excellent antifungal target.     

The effect of pH on the conversion of superoxide to hydroxyl free radicals

The conversion of superoxide (O-.2) to the hydroxyl (HO.) free radical by superoxide-driven Fenton reactions was measured by the formation of hydroxylated derivatives from benzoate. Among a range of catalysts required for the conversion, the Fe3+EDTA complex was the most effective. The effect of superoxide dismutase and catalase indicated that O-.2 and H2O2 were essential reactants, while the formation of authentic HO. was confirmed by the inhibiting capacities of formate, t-butanol, and mannitol. The conversion of O-.2 to HO. was tested over a broad pH range, and was found to be highest at pH 4.8 whether Fe3+EDTA or free Fe3+ were used as the catalysts. When Fe3+EDTA was used at the optimum pH, every HO. produced required 3.7 O-.2 radicals, close to the theoretical limit of one HO. from every three O-.2 radicals generated.     

Enzymatic conversion of dihydrouracil into uracil

We found that some strains of Rhodotorula glutinis can oxideze dihydrourcil to uracil, and we converted dihydrouracil into uracil using the resting and immobilized cells of R. glutinis IFO-1389.The optimum pH of the conversion of dihydrouracil into uracil was 7.8. Oxygen supply was essential to the conversion. With resting cells, the addition of both o-phenanthroline and Triton X-100 caused increase of the yield of uracil about ten times as much as that with no addition.The conversion ratios of dihydrouracil into uracil using immobilized-cell beds, which were made with chitosan and glutaraldehyde, were 100, 98, and 77% when the concentration of dihydrouracil were 1, 2, and 3 (w/v)%, respectively, for 68 h at 30°C.     

Some studies of crosslinking chitosan-glutaraldehyde interaction in a homogeneous system.

Chitosan dissolved in acetic acid reacted with glutaraldehyde solution, ranging in concentration from 0.10 to 25.0 x 10(-2) mol dm3. The modified polymers were characterized by means of carbon, hydrogen and nitrogen elemental analysis, scanning electron microscopy, X-ray diffractometry, 13C nuclear magnetic resonance (NMR), infrared and Raman spectroscopies. The uptake of metallic cations in aqueous medium was checked through copper. The obtained data from 13C NMR, infrared and Raman spectroscopies evidenced the formation of an ethylenic double bond in the chitosan glutaraldehyde interaction. These data suggest that free pendant amine groups of chitosan polymer interact with the aldehydic group of the glutaraldehyde to form stable imine bonds, due to the resonance established with adjacent double ethylenic bonds. The crosslinking is formed by the nonuniform length of chains and by terminal unities. The crosslinking formation can involve two chitosan unities belonging, or not, to the same polymeric chain. The sequence of reactions was established for a chitosan:glutaraldehyde molar proportion of 1:20. The degree of crystallinity and particle size decreased as the amount of glutaraldehyde was increased in the polymer. Physical and chemical properties are not just affected for the chitosan glutaraldehyde reaction, but are also affected strongly by the dissolution of the natural chitosan.     

Relevance of molecular weight of chitosan and its derivatives and their antioxidant activities in vitro

The antioxidant potency of different molecular weight (DMW) chitosan and sulfated chitosan derivatives was investigated employing various established in vitro systems, such as superoxide (O(2)(.-))/hydroxyl ((-.)OH) radicals scavenging, reducing power, iron ion chelating. As expected, we obtained several satisfying results, as follows: firstly, low molecular weight chitosan had stronger scavenging effect on O(2)(.-) and (-.)OH than high molecular weight chitosan. For example the O(2)(.-) scavenging activity of low molecular weight chitosan (9 kDa) and high molecular weight chitosan (760 kDa) were 85.86% and 35.50% at 1.6 mg/mL, respectively. Secondly, comparing with DMW chitosan, DMW sulfated chitosans had the stronger inhibition effect on O(2)(.-). At 0.05 mg/mL, the scavenging activity on O(2)(.-) reached 86.26% for low molecular weight chitosan sulfate (9 kDa), but that of low molecular weight chitosan (9 kDa) was 85.86% at 1.6 mg/mL. As concerning chitosan and sulfated chitosan of the same molecular weight, scavenging activities of sulfated chitosan on superoxide and hydroxyl radicals were more pronounced than that of chitosan. Thirdly, low molecular weight chitosan sulfate had more effective scavenging activity on O(2)(.-) and (-.)OH than that of high molecular weight chitosan sulfate. Fourthly, DMW chitosans and sulfated chitosans were efficient in the reducing power, especially LCTS. Their orders were found to be LCTS>CTS4>HCTS>CTS3>CTS2>CTS1>CTS. Fifthly, CTS4 showed more considerable ferrous ion-chelating potency than others. Finally, the scavenging rate and reducing power of DMW chitosan and sulfated derivatives increased with their increasing concentration. Moreover, change of DMW sulfated chitosans was the most pronounced within the experimental concentration. However, chelating effect of DMW chitosans were not concentration dependent except for CTS4 and CTS1.     

Effects of Spray Drying on Physicochemical Properties of Chitosan Acid Salts

The effects of spray-drying process and acidic solvent system on physicochemical properties of chitosan salts were investigated. Chitosan used in spray dryings was obtained by deacetylation of chitin from lobster (Panulirus argus) origin. The chitosan acid salts were prepared in a laboratory-scale spray drier, and organic acetic acid, lactic acid, and citric acid were used as solvents in the process. The physicochemical properties of chitosan salts were investigated by means of solid-state CP-MAS ¹³C nuclear magnetic resonance (NMR), X-ray powder diffraction (XRPD), differential scanning calorimetry, and Fourier transform infrared spectrometry (FTIR) and near-infrared spectroscopy. The morphology of spray-dried chitosan acid salts showed tendency toward higher sphericity when higher temperatures in a spray-drying process were applied. Analysis by XRPD indicated that all chitosan acid salts studied were amorphous solids. Solid-state ¹³C NMR spectra revealed the evidence of the partial conversion of chitosan acetate to chitin and also conversion to acetyl amide form which appears to be dependent on the spray-drying process. The FTIR spectra suggested that the organic acids applied in spray drying may interact with chitosan at the position of amino groups to form chitosan salts. With all three chitosan acid salts, the FTIR bands at 1,597 and 1,615 cm⁻¹ were diminished suggesting that –NH groups are protonated. The FTIR spectra of all chitosan acid salts exhibited ammonium and carboxylate bands at 1,630 and 1,556 cm⁻¹, respectively. In conclusion, spray drying is a potential method of preparing acid salts from chitosan obtained by deacetylation of chitin from lobster (P. argus) origin.     

A new approach for extracellular spin trapping of nitroglycerin-induced superoxide radicals both in vitro and in vivo

Anti-ischemic therapy with nitrates is complicated by the induction of tolerance that potentially results from an unwanted coproduction of superoxide radicals. Therefore, we analyzed the localization of in vitro and in vivo, glyceryl trinitrate (GTN)-induced formation of superoxide radicals and the effect of the antioxidant vitamin C and of superoxide dismutase (SOD). Sterically hindered hydroxylamines 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CP-H) and 1-hydroxy-4-phosphonooxy-2,2,6,6-tetramethylpiperidin (PP-H) can be used for in vitro and in vivo quantification of superoxide radical formation. The penetration/incorporation of CP-H or PP-H and of their corresponding nitroxyl radicals was examined by fractionation of the blood and blood cells during a 1-h incubation. For monitoring in vivo, GTN-induced (130 microg/kg) O2*- formation CP-H or PP-H were continuously infused (actual concentration, 800 microM) for 90 to 120 min into rabbits. Formation of superoxide was determined by SOD- or vitamin C-inhibited contents of nitroxide radicals in the blood from A. carotis. The incubation of whole blood with CP-H, PP-H, or corresponding nitroxyl radicals clearly shows that during a 1-h incubation, as much as 8.3% of CP-H but only 0.9% of PP-H is incorporated in cytoplasm. Acute GTN treatment of whole blood and in vivo bolus infusion significantly increased superoxide radical formation as much as 4-fold. Pretreatment with 20 mg/kg vitamin C or 15,000 U/kg superoxide dismutase prevented GTN-induced nitroxide formation. The decrease of trapped radicals after treatment with extracellularly added superoxide dismutase or vitamin C leads to the conclusion that GTN increases the amount of extracellular superoxide radicals both in vitro and in vivo.     

C5'- and C3'-sugar radicals produced via photo-excitation of one-electron oxidized adenine in 2'-deoxyadenosine and its derivatives

We report that photo-excitation of one-electron-oxidized adenine [A(-H)•] in dAdo and its 2′-deoxyribonucleotides leads to formation of deoxyribose sugar radicals in remarkably high yields. Illumination of A(-H)• in dAdo, 3′-dAMP and 5′-dAMP in aqueous glasses at 143 K leads to 80-100% conversion to sugar radicals at C5′ and C3′. The position of the phosphate in 5′- and 3′-dAMP is observed to deactivate radical formation at the site of substitution. In addition, the pH has a crucial influence on the site of sugar radical formation; e.g. at pH ~5, photo-excitation of A(-H)• in dAdo at 143 K produces mainly C5′• whereas only C3′• is observed at high pH ~12. ¹³C substitution at C5′ in dAdo yields ¹³C anisotropic couplings of (28, 28, 84) G whose isotropic component 46.7 G identifies formation of the near planar C5′•. A β-¹³C 16 G isotropic coupling from C3′• is also found. These results are found to be in accord with theoretically calculated ¹³C couplings at C5′ [DFT, B3LYP, 6-31(G) level] for C5′• and C3′•. Calculations using time-dependent density functional theory [TD-DFT B3LYP, 6-31G(d)] confirm that transitions in the near UV and visible induce hole transfer from the base radical to the sugar group leading to sugar radical formation.     

Radical formation in single crystals of hypoxanthine.HCl.H2O, inosine, and the disodium salt of 5'-inosine-monophosphate.

Radical formation in single crystals of hypoxanthine.HCl.H2O, inosine and Na2-5'-IMP.(7.5 H2O) by X-irradiation has been studied using electron-spin-resonance spectroscopy at 9.5 and 35 GHz. In all crystals both H-addition radicals at position C2 and C8 of the purine ring are found. The coupling constants of these two radicals are different and depend strongly on the protonation state of the base. INDO-calculations indicate that the C8-radical is protonated at O6. In Na2-5'-IMP OH-addition radicals at position C2 of the purine ring are formed. Electron adduct radicals are found in the neutral and the N7-protonated base after X-irradiation at 77 K. In Na2-5'-IMP no electron adduct is formed but a radical which probably is the cation. In hypoxanthine.HCl.H2O a radical could be observed after X-irradiation at 77 K, which results from addition of a Cl- to the nitrogen N1.     

Chitosan bicomponent nanofibers and nanoporous fibers

Nanofibers with average diameters between 20 and 100nm have been prepared by electrospinning of 82.5% deacetylated chitosan (Mv=1600 kDa) mixed with poly(vinyl alcohol) (PVA, Mw=124-186 kDa) in 2% (v/v) aqueous acetic acid. The formation of bicomponent fibers was feasible with 3% concentration of solution containing up to an equal mass of chitosan. Finer fibers, fewer beaded structures and more efficient fiber formation were observed with increasing PVA contents. Nanoporous fibers could be generated by removing the PVA component in the 17/83 chitosan/PVA bicomponent fibers with 1M NaOH (12 h). Fiber formation efficiency and composition uniformity improved significantly when the molecular weight of chitosan was halved by alkaline hydrolysis (50 wt% aqueous NaOH, 95 degrees C, 48 h). The improved uniform distribution of chitosan and PVA in the bicomponent fibers was attributed to better mixing mostly due to the reduced molecular weight and to the increased deacetylation of the chitosan.     

Hyperoxia and xanthine dehydrogenase/oxidase activities in rat lung and heart

Cell injury from hyperoxia is associated with increased formation of superoxide radicals (O2-). One potential source for O2- radicals is the reduction of molecular O2 catalyzed by xanthine oxidase (XO). Physiologically, this reaction occurs at a relatively low rate, because the native form of the enzyme is xanthine dehydrogenase (XD) which produces NADH instead of O2-. Reports of accelerated conversion of XD to XO, and increased formation of O2- formation in ischemia-reperfusion injury, led us to examine whether hyperoxia, which is known to increase O2- radical formation, is associated with increased lung XO activity, and accelerated conversion of XD to XO. We exposed 3-month-old rats either to greater than 98% O2 or room air. After 48 h, we sacrificed the rats and measured XD and XO activities and uric acid contents of the lungs. We also measured the activities of the two enzymes in the heart as a control organ. We found that the activity of XD was not altered significantly by hyperoxia in rat lungs or hearts, but XO activity was markedly lower in the lung, whether expressed per whole organ or per milligram protein, and remained unchanged in the heart. Lung uric acid content was also significantly lower with hyperoxia. The decrease in lung XO activity may reflect inactivation of the enzyme by reactive O2 metabolites, possibly as a negative feedback mechanism. The concomitant decrease in uric acid content suggests either decreased production mediated by XO due to its inactivation or greater utilization of uric acid as an antioxidant. We examined these postulates in vitro using a xanthine/xanthine oxidase system and found that H2O2, but not uric acid, has an inhibitory effect on O2- formation in the system. We therefore conclude that hyperoxia is not associated with increased conversion of XD to XO, and that the exact contribution of XO to hyperoxic lung injury in vivo remains unclear.     

Factors affecting the free radical scavenging behavior of chitosan sulfate

Scavenging activity of hydroxyethyl chitosan sulfate (HCS) against 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl and carbon-centered radical species were studied using electron spin resonance (ESR) spectroscopy. In addition, its antioxidant activity to retard lipid peroxidation was also evaluated in a linoleic acid model system. HCS could scavenge DPPH (33.78%, 2.5 mg/mL) and carbon-centered radicals (67.74%, 0.25 mg/mL) effectively. However, chitosan sulfate did not exhibit any scavenging activity against hydroxyl radicals, but increased its generation. This was different from the published literature and was presumed due to the loss of chelating ability on Fe2+. This assumption could further confirm from the results obtained for Fe2+-ferrozine method that upon sulfation chitooligosaccharides lost its chelation properties. Therefore, HCS can be identified as antioxidant that effectively scavenges carbon centered radicals to retard lipid peroxidation.     

Peroxidase catalysed oxygen activation by arylamine carcinogens and phenol

Peroxidase catalysed the formation of active oxygen in the presence of NADH or GSH and traces of H2O2 and arylamine or phenolic substrates. Some oxygen activation occurred with some arylamines even in the absence of NADH or GSH. Oxygen consumption was proportional to the NADH oxidized or GSSG formed. Approximately 0.80 and 0.40 mol of oxygen were consumed per mole of NADH or GSH oxidized respectively. The requirement for trace amounts of hydrogen peroxide and arylamine or phenolic substrates suggest that redox cycling resulted in H2O2 formation. It is proposed that initially formed phenoxy radicals or arylamine cation radicals oxidize NADH or GSH to radicals which react with oxygen to form superoxide radicals and H2O2.