Hiroe Yoshioka

Interaction of (+)-Catechin with a Lipid Bilayer Studied by the Spin Probe Method

The interaction of (+)-catechin with a lipid bilayer was examined by the spin probe method. The spin probe, 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), was dissolved in an aqueous dipalmitoylphosphatidylcholine (DPPC) dispersion containing (+)-catechin. The temperature dependence of the TEMPO parameter was measured. The increase of this parameter due to pretransition was eliminated by the addition of (+)-catechin, suggesting that it was adsorbed to the lipid membrane surface in the gel state, which hindered the change of the membrane from a flat to wavy structure. In the temperature region of the main transition, the TEMPO parameter increased rapidly, then gradually with increasing temperature, which could be explained by the eutectic phase diagram. The rotational correlation time of a spin probe 16-doxylstearic acid and the order parameter of 5-doxylstearic acid in the aqueous dispersion system of egg yolk phosphatidylcholine revealed that the motion of the alkyl chain in the liquid crystal state was hindered in the center of the membrane as well as near the surface by the adsorption of (+)-catechin.

Kinetic analysis of the effect of (-)-epigallocatechin gallate on the DNA scission induced by Fe(II).

The DNA strand scission induced by Fe(II) in a citrate buffer solution and the effect of (−)-epigallocatechin gallate (EGCg) were kinetically analyzed. The rate of consumption of dissolved oxygen by Fe(II) in each of these solutions was measured and paralleled that DNA scission. Coordinated EGCg accelerated these reactions. Curves of the time-course characteristics of DNA scission were simulated by using the rate constant of oxygen consumption and by assuming that scission with the hydroxyl radical (OH), which was formed from the dissolved oxygen, proceeded competitively with the scavenging of OH by citrate, Cl− ions and EGCg added. Free EGCg acted as a DNA scission inhibitor to scavenge OH, in contrast to the case of the coordinated one. This analysis is useful for estimating the rate constant of the reaction between an antioxidant and OH, and might provide a new method for measuring the OH-scavenging activity.

A novel method of measuring hydroxyl radical-scavenging activity of antioxidants using γ-irradiation

A new method using ESR spin trapping was proposed for measuring the scavenging activity of antioxidants for the hydroxyl (OH) radical. (-)-Epigallocatechin gallate (EGCg) and 5,5-dimethyl-1-pyrrolline N-oxide (DMPO) were used as the antioxidant and spin trapping agent, respectively. The conventional method using a Fenton reaction had problems associated with the estimation of activity, because the antioxidant disturbs the system for generating OH radical by coordinating on Fe2+ and by consuming H2O2, besides scavenging the spin adduct (DMPO-OH). Intense γ-irradiation was therefore used to generate OH radicals, and the intensity decrease in DMPO-OH after irradiation was followed to obtain the rate constant for the scavenging of DMPO-OH by EGCg. The intensities were extrapolated to zero time to estimate the quantity of DMPO-OH formed during γ-irradiation. By using these values, the reaction rate constant between OH radical and EGCg was calculated as a ratio to that of DMPO. It was shown that this method is useful for comparing the OH radical-scavenging activity of various antioxidants.

Determination of trace amounts of thallium in commercial radioactive 204Tl samples by the redox sub-superequivalence method of isotope dilution analysis

Trace amounts of thallium in a commercial radioactive 204Tl sample were determined using the sub-superequivalence method of isotope dilution analysis (SSE-IDA) and sub-stoicheiometric isotope dilution analysis (Subst-IDA), and the results obtained were compared. Before the determination, various conditions for oxidation and extraction were examined. An HCl-HClO4 mixture was the best solvent for oxidation by KBrO3 and 20% tributyl phosphate in benzene was suitable for the separation of TlIII. Under conditions where ordinary Subst-IDA gives large errors, accurate determinations with errors of a few per cent. were possible with SSE-IDA. The volume of the reaction solution was minimised in order to reduce the radioactive waste problem.

Detection of 2-deoxy-D-ribose radicals generated by the reaction with the hydroxyl radical using a rapid flow-ESR method.

ESR spectrum of the short-lived radicals derived from 2-deoxy-D-ribose by the reaction with the hydroxyl radical (HO·) was measured using a rapid flow method. A dielectric mixing resonator was used for the measurement, which made it possible to measure the highly sensitive ESR spectra of the radicals with a lifetime of the order of milliseconds. A complex spectrum was obtained and the spectral simulation was done to show that it was the superposition of the signals due to five radicals (I-V). Three of them were those formed by the dehydrogenation with the HO· at C-1 (I), C-3 (II), and C-4 (III) positions of the 2-deoxy-D-ribose molecule. The other two (IV and V) were carbonyl-conjugated radicals formed by the elimination of a water molecule from III and II. The results showed that dehydrogenation occurred randomly at the positions where hydroxyl groups are attached, but the most preferred position was C-3 and the radical position moved from C-3 to C-4 by the elimination of water molecule.

Scavenging activity of tea catechins on hydroxyl radical formed in tritiated water studied by the solid-state spin trapping method

ABSTRACT Solid state spin trapping method was applied for measuring the scavenging activity of hydroxyl (OH) radical formed by the beta ray from tritiated water with four tea catechins, (−)-epicatechin (EC), (−)-epicatechin gallate (ECg), (−)-epigallocatechin (EGC) and (−)-epigallocatechin gallate (EGCg). The activity was in the order of EGCg > ECg > EGC > EC. This is consistent with the order of the number of phenolic hydroxyl groups in each catechin molecule. However, it was shown that EGCg had stronger activity than others, which suggested the interaction between the B ring and the gallate group.

Determination of trace amounts of tellurium by the sub-superequivalence method of isotope dilution analysis

Trace amounts of tellurium in synthetic samples were determined by the redox sub-superequivalence method of isotope dilution analysis (SSE-IDA) and the results were compared with those of sub-stoichiometric isotope dilution analysis (Subst-IDA) in order to demonstrate the usefulness of SSE-IDA. 125mTeIV was separated from an HCl solution in which 125Sb and 125mTeIV were in radioactive equilibrium and 125mTe was used as a tracer. The reactant (TeIV) was oxidized sub-stoichiometrically with a solution of K2Cr2O7 in 0.1 mol dm–3 HCl, followed by the extraction of TeIV with 20% tributyl phosphate–kerosene solution (after changing the concentration of HCl to 5 mol dm–3) in order to separate TeIV and TeVI. Activities of TeVI in each aqueous phase were measured and the amount of tellurium was determined according to the graphical method of SSE or Subst-IDA. Under the conditions where Subst-IDA gives large errors, accurate determinations were possible using SSE-IDA with errors of less than 5–6 per cent. The limit of the determination was about 2 µg ml–1.