Yoshimi Sueishi

An oxygen radical absorbance capacity-like assay that directly quantifies the antioxidant's scavenging capacity against AAPH-derived free radicals.

A new method is proposed for the evaluation of oxygen radical absorbance capacity (ORAC). The current fluorescence-based ORAC assay (ORAC-FL) is an indirect method that monitors the antioxidants ability to protect the fluorescent probe from free radical-mediated damage, and an azo-radical initiator, AAPH (2,2-azobis(2-amidinopropane) dihydrochloride), has been used as a thermal free radical source. The new ORAC assay employs a short in situ photolysis of AAPH to generate free radicals. The electron paramagnetic resonance (EPR) spin trapping method was employed to identify and quantify AAPH radicals. In the presence of antioxidant, the level of AAPH radicals was decreased, and ORAC-EPR values were calculated following a simple kinetic formulation. Alkyl-oxy radical was identified as the sole decomposition product from AAPH; therefore, we concluded that ORAC-FL is the assay equivalent to alkyl-oxy radical scavenging capacity measurement. ORAC-EPR results for several antioxidants and human serum indicated that the overall tendency is in agreement with ORAC-FL, but absolute values showed significant discrepancies. ORAC-EPR is a rapid and simple method that is especially suitable for thermally labile biological specimens because the sample heating is not required for free radical production.

Nitric oxide (NO) scavenging capacity of natural antioxidants.

Nitric oxide (NO)-scavenging capacities of several hydrophilic antioxidants were determined by using the PTIO method, a competitive NO-scavenging method with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). Relative NO-scavenging rates of antioxidants were measured with respect to PTIO and the scavenging rate constants were calculated based on PTIOs rate constant. Results indicated that NO-scavenging rate constants of the antioxidants were: uric acid (2.5)>caffeic acid (1.2)>trolox (1.0)>genistein (0.19)>glutathione (0) ≈N-acetylcysteine (0), where the numbers are expressed in trolox equivalent unit. The oxidation potentials of these antioxidants were measured and the order in the magnitude of oxidation potential was in good accordance with NO-scavenging capacity. Based on the results, we have suggested that the primary chemical process of the antioxidant reaction with NO can be characterised with the electron transfer from NO to the antioxidant.

Suppression of NO production and 8-nitroguanosine formation by phenol-containing endocrine-disrupting chemicals in LPS-stimulated macrophages: Involvement of estrogen receptor-dependent or -independent pathways

Since the endocrine and immune systems share portions of some intracellular signaling pathways, endocrine-disrupting chemicals (EDCs) are considered potential agents for influencing inflammatory responses. Here, we investigated the effect of EDCs on lipopolysaccharide (LPS)-induced NO production and NF-kappaB activation in the RAW264.7 mouse macrophage cell line. Five phenol-containing EDCs were investigated, namely bisphenol A (BPA), the alkyl phenols p-n-nonylphenol (NP) and p-n-octylphenol (OP), and the chlorinated phenols 2,4-dichlorophenol (DCP) and pentachlorophenol (PCP). Our results revealed that these chemicals dose-dependently suppressed LPS-induced NO production, as reflected by decreased NO(x) content. The suppressive effects of BPA, NP and OP, but not PCP or DCP, were blocked by the estrogen receptor (ER) inhibitor, ICI182780. ELISA-based quantification of the DNA-binding activity of free p65 NF-kappaB showed that LPS-induced NF-kappaB activation was significantly diminished by EDC treatment. Furthermore, immunocytochemical analysis of 8-nitroguanosine, a unique index of NO-mediated signaling, showed that 8-nitroguanosine formation increased in LPS-stimulated cells, but this increase was inhibited by the tested EDCs. These results demonstrate that EDCs suppress NO production and NF-kappaB activation in LPS-stimulated macrophages through ER-dependent (BPA, NP, OP) and -independent (PCP, DCP) pathways. The EDCs further inhibited 8-nitroguanosine formation, suggesting that they interfere with NO-mediated signaling. Thus, EDCs might play important roles in the inflammatory response and host defense system against foreign pathogens.

Scavenging rate constants of hydrophilic antioxidants against multiple reactive oxygen species

Scavenging rate constants of eight hydrophilic antioxidants, including caffeic acid, chlorogenic acid, genistein, glutathione, N-acetylcysteine, rutin, trolox, and uric acid against multiple ROS, namely superoxide anion, hydroxyl radical, singlet oxygen, and alkoxyl radical were determined with the electron spin resonance method. Direct flash photolysis measurement of the second-order rate constant in the reaction of alkoxyl radical plus the spin trap 5,5-dimethyl-pyrroline N-oxide made it possible to evaluate scavenging rate constants in antioxidants. The magnitudes of scavenging rate constants were notably dependent on the character of each ROS and the overall rate constants were highest in hydroxyl radical scavenging and the lowest in superoxide anion. The highest scavenging rate constant against superoxide anion was obtained by chlorogenic acid (2.9 × 105 M−1 s−1) and the lowest was by N-acetylcysteine (5.0 × 102 M−1 s−1). For singlet oxygen, the highest was by glutathione (9.4 × 108 M−1 s−1) and the lowest was by uric acid (2.3 × 106 M−1 s−1). All other numbers are listed and illustrated. Redox potential measurements of the antioxidants indicated that the antioxidants are likely to react with superoxide anion and singlet oxygen through electron transfer processes.

Oxygen radical absorbance capacity (ORAC) of cyclodextrin-solubilized flavonoids, resveratrol and astaxanthin as measured with the ORAC-EPR method

Recently, we proposed an oxygen radical absorbance capacity method that directly quantifies the antioxidant’s scavenging capacity against free radicals and evaluated the radical scavenging abilities for water soluble antioxidant compounds. In this study, we determined the radical scavenging abilities of lipophilic antioxidants which were solubilized by cyclodextrin in water. Commonly employed fluorescence-based method measures the antioxidant’s protection capability for the fluorescent probe, while we directly quantify free-radical level using electron paramagnetic resonance spin trapping technique. In addition, the spin trapping-based method adopted controlled UV-photolysis of azo-initiator for free radical generation, but in fluorescence-based method, thermal decomposition of azo-initiator was utilized. We determined the radical scavenging abilities of seven well-known lipophilic antioxidants (five flavonoids, resveratrol and astaxanthin), using methylated β-cyclodextrin as a solubilizer. The results indicated that the agreement between spin trapping-based and fluorescence-based values was only fair partly because of a large variation in the previous fluorescence-based data. Typical radical scavenging abilities in trolox equivalent unit are: catechin 0.96; epicatechin 0.94; epigallocatechin gallate 1.3; kaempferol 0.37; myricetin 3.2; resveratrol 0.64; and astaxanthin 0.28, indicating that myricetin possesses the highest antioxidant capacity among the compounds tested. We sorted out the possible causes of the deviation between the two methods.

Effects of Substituent and Solvent on Inclusion Complexation of β-Cyclodextrins with Azobenzene Derivatives

The equilibrium constants (K) for the inclusion complex formationof β-cyclodextrin (β-CD) with Methyl Orange (MO) and substituted azoanilinium chlorides were determinedspectrophotometrically. Based on the results, the substituent effecton the inclusion complexation of β-CD with azoanilinium chlorides was discussed indetail. Further, the solvent effects on the inclusion complexation of MO with β-CD andheptakis(2,6-di-O-methyl)-β-cyclodextrin (DM-β-CD) wereexamined in aqueous organic mixtures with water-miscible organic compounds(dimethylsulfoxide, acetonitrile, N,N-dimethylformamide, and acetone). It was found that the K value for the inclusion complexformation with β-CD and DM-β-CD decreases remarkably with increasing ratioof organic solvents, dependent of the surface tension of solvent mixtures.

A multiple free-radical scavenging (MULTIS) study on the antioxidant capacity of a neuroprotective drug, edaravone as compared with uric acid, glutathione, and trolox.

Edaravone (3-methyl-1-phenyl-2-pyrazoline-5-one) is a neuroprotective drug that has been used for brain ischemia injury treatment. Because its activity is speculated to be due to free radical scavenging activity, we carried out a quantitative determination of edaravones free radical scavenging activity against multiple free radical species. Electron spin resonance (ESR) spin trapping-based multiple free-radical scavenging (MULTIS) method was employed, where target free radicals were hydroxyl radical, superoxide anion, alkoxyl radical, alkylperoxyl radical, methyl radical, and singlet oxygen. Edaravone showed relatively high scavenging abilities against hydroxyl radical (scavenging rate constant k=2.98×10(11) M(-1) s(-1)), singlet oxygen (k=2.75×10(7) M(-1) s(-1)), and methyl radical (k=3.00×10(7) M(-1) s(-1)). Overall, edaravones scavenging activity against multiple free radical species is as robust as other known potent antioxidant such as uric acid, glutathione, and trolox. A radar chart illustration of the MULTIS activity relative to uric acid, glutathione, and trolox indicates that edaravone has a high and balanced antioxidant activity with low specificity.

Serum Hydroxyl Radical Scavenging Capacity as Quantified with Iron-Free Hydroxyl Radical Source

We have developed a simple ESR spin trapping based method for hydroxyl (OH) radical scavenging-capacity determination, using iron-free OH radical source. Instead of the widely used Fenton reaction, a short (typically 5 seconds) in situ UV-photolysis of a dilute hydrogen peroxide aqueous solution was employed to generate reproducible amounts of OH radicals. ESR spin trapping was applied to quantify OH radicals; the decrease in the OH radical level due to the specimen’s scavenging activity was converted into the OH radical scavenging capacity (rate). The validity of the method was confirmed in pure antioxidants, and the agreement with the previous data was satisfactory. In the second half of this work, the new method was applied to the sera of chronic renal failure (CRF) patients. We show for the first time that after hemodialysis, OH radical scavenging capacity of the CRF serum was restored to the level of healthy control. This method is simple and rapid, and the low concentration hydrogen peroxide is the only chemical added to the system, that could eliminate the complexity of iron-involved Fenton reactions or the use of the pulse-radiolysis system.

A volumetric study on the thermal cis-to-trans isomerization of 4-(dimethylamino)-4′-nitroazobenzene and 4,4′-bis(dialkylamino)azobenzenes: evidence of an inversion mechanism

The reaction volume (ΔV) for the thermal cis-to-trans isomerization of 4-(dimethylamino)-4′-nitroazobenzene in various solvents has been obtained by means of the photostationary state and capillary method. It has been shown that the activation volume (ΔV‡) is closely related to the reaction volume in each solvent. Substantial kinetic pressure and solvent effects have been observed for 4,4′-bis(dimethylamino)azobenzene, 4,4′-bis(diethylamino)azobenzene, and 4,4′-(dipyrrolidin-1-yl)azobenzene. The π–π* conjugation band of the trans-isomers of these compounds shows a remarkable solvatochromic shift, and logarithms of kinetic constants in various solvents are satisfactorily correlated with the Taft π* scale, which represents solvent polarity–polarizability interactions. From McRaes formula, it has been deduced that the interaction between the solvent dipole and the solvent-induced dipole has an important influence on the behaviour of these ‘push–pull’ and ‘push–push’ azobenzenes. The results afford unequivocal evidence that the isomerization proceeds via a coplanar inversion transition state which is electronically similar to the trans-isomer.

Complexation of phenols with β- and γ-cyclodextrins: determination of the association constants by using the isomerization of spiropyran

As a chemical indicator, the isomerization rate of so-called 6-SO3−-spiropyran was used to estimate the equilibrium constants for the inclusion complex formation of β- and γ-cyclodextrins (β and γ-CDs). The association constants for inclusion complexes of β- and γ-CDs with various kinds of phenols were determined. It was found that β- and γ-CDs form 1:1 and 1:2 inclusion complexes with phenols, respectively. The substituent effects on the association constants for the 4-substituted phenols–β-CD system can be interpreted in terms of the geometry of the inclusion complex. The magnitude of the association constants for the inclusion complex is related to molecular polarizability of the guest molecule. Based on the results, the dominant factor for CD complexation with phenols is discussed. Copyright