Finding of Remarkable Synergistic Effect on the Aroxyl-Radical-Scavenging Rates (ks) under the Coexistence of Vitamin E Homologues (or Vegetable Oils) and Ubiquinol-10: Proposal of A New Mechanism to Explain An Increase of ks Value.

Measurements of aroxyl (ArO · )-radical-scavenging rate constants (ksAOH) of antioxidants (AOHs) (i.e., α-, ß-, γ-, δ-Tocopherol (TocH) and ubiquinol-10 (UQ10H2)) were performed in ethanol/chloroform/H2O (50/50/1, v/v) solution, using stopped-flow spectrophotometry. ksAOH values were measured not only for each AOH, but also for the mixtures of two AOHs (i.e., TocH and UQ10H2). ksTocH values for α-, ß-, γ-, δ-TocH increased 1.21, 1.28, 1.55, and 1.19 times, respectively, under the coexistence of constant concentrations of UQ10H2. Similar measurements were performed for eight vegetable oils 1 - 8, containing different concentrations of α-, ß-, γ-, δ-tocopherol (TocH) and -tocotrienol (Toc-3H). ksOil values of all eight vegetable oils 1 - 8 also increased 1.24 - 1.54 times under the coexistence of constant concentrations of UQ10H2. A new mechanism to explain the notable increase of ksAOH values under the coexistence of two kinds of phenolic AOHs was proposed. UV-vis absorption of α-, ß-, γ-Toc · radicals, produced by reaction of α-, ß-, γ-TocHs (or vegetable oils 1 - 8) with ArO · , disappeared under the coexistence of TocHs (or oils) and UQ10H2, suggesting that the prooxidant reaction resulting from the presence of Toc · radicals is suppressed in the presence of UQ10H2.

Kinetic Study of Singlet-Oxygen Quenching and Aroxyl-Radical Scavenging Activities of Vitamin E Homologs and Fatty Acids Present in Vegetable Oils.

Recently, singlet-oxygen (1O2) quenching and aroxyl-radical (ArO·) scavenging rates (kQ and kS, respectively) of eight vegetable oils were measured in the ethanol/chloroform/D2O solution. Furthermore, the kQ and kS values and concentrations of four tocopherols and four tocotrienols contained in the vegetable oils were measured. In this study, the concentrations of nine fatty acids (including stearic, oleic, linoleic, and linolenic acids) comprising the above-mentioned eight vegetable oils were determined by gas chromatography. The kQ and kS values for ethyl stearate, ethyl oleate, ethyl linoleate, methyl linolenate, and glyceryl trioleate in the ethanol/chloroform/D2O solution were measured by UV-vis spectrophotometry. Based on the results obtained for the above-mentioned fatty acid esters, the kQ and kS values were estimated for nine fatty acids. Furthermore, comparisons of kQ values observed for the vegetable oils with the sum of the product {∑kQAO-i [AO-i]} of the kQAO-i values obtained for each antioxidant-i (AO-i) and the concentrations ([AO-i]) of AO-i (i.e., four tocopherols (& four tocotrienols) and nine fatty acids) contained in vegetable oils were performed. Based on the results, a detailed comparison of the contributions of the tocopherols (and tocotrienols) and the fatty acids to the 1O2-quenching rate constants (kQ) was performed. This indicated that both the tocopherols (and tocotrienols) and the fatty acids contribute to the 1O2- quenching. A similar comparison was conducted for the ArO· -scavenging rate constants (kS). The results suggested that only the tocopherols (and tocotrienols) contained in the oils contributed to the ArO· -scavenging, with negligible contribution from the fatty acids.

Antioxidant Activity of Foods: Development of Singlet Oxygen Absorption Capacity (SOAC) Assay Method.

Recently a new assay method that can quantify the singlet oxygen absorption capacity (SOAC) of antioxidants (AOs) and food extracts was proposed. Singlet oxygen (1O2) quenching rates (kQ) and the relative SOAC values were measured for many carotenoids and phenolic AOs in ethanol/chloroform/D2O (50 : 50 : 1, v/v/v) solution at 35ºC using UV-vis spectrophotometry. It has been clarified that the SOAC method is useful to evaluate the 1O2-quenching activity of lipophilic and hydrophilic AOs having 5 orders of magnitude different rate constants (kQ). Measurements of the kQ and SOAC values were also performed for 39 kinds of food extracts. The results indicate that the SOAC method is useful to evaluate the 1O2-quenching activity of food extracts having two orders of magnitude different kQ values. Further, the kQ values for the reaction of 1O2 with 8 carotenoids and 8 vitamin E homologues were measured in an aqueous Triton X-100 (5.0 wt %) micellar solution (pH 7.4). Results obtained demonstrate that the kQ values of AOs in homogeneous and heterogeneous solutions vary notably depending on (i) polarity (dielectric constant (ε)) of the reaction field between AOs and 1O2, (ii) local concentration of AOs, and (iii) mobility of AOs in solution. Measurements of kQ and SOAC values in a micellar solution may be useful for evaluating the 1O2-quenching activity of AOs in biological systems. Furthermore, measurements of the SOAC values were performed for 32 kinds of food extracts using a microplate reader. The SOAC assay method was validated by inter-laboratory validation study due to 14 laboratories.

Kinetic Study of the Quenching Reaction of Singlet Oxygen by Eight Vegetable Oils in Solution.

A kinetic study of the reaction of singlet oxygen (1O2) with eight vegetable oils 1-8 containing different concentrations of tocopherols (Tocs) and tocotrienols (Toc-3s) was performed. The second-order rate constants (kQ) for the reaction of 1O2 with vegetable oils 1-8 (rice bran, perilla, rape seed, safflower, grape seed, sesame, extra virgin olive, and olive oils) were measured in ethanol/chloroform/D2O (50:50:1, v/v/v) solution at 35°C using UV-vis spectrophotometry. Furthermore, comparisons of kQ values determined for the above oils 1-8 with the sum of the product {∑kQAO-i [AO-i]/105} of the kQAO-i values obtained for each antioxidant (AO-i) and concentration (in mg/100 g) ([AO-i]/105) of AO-i (Tocs and Toc-3s) contained in the oils 1-8 were performed. The observed kQ values were not reproduced by the kQ values calculated using only the concentrations of the four Tocs and Toc-3s. These results suggest that the contribution of fatty acids contained in the oils 1-8 is also necessary to fully explain the kQ values. Recently, the second-order rate constants (kS) for the reaction of aroxyl radical (ArO・) with the same vegetable oils 1-8 were measured in the same solvent at 25℃ using stopped-flow spectrophotometry (Ref. 23). The kS values obtained could be well explained as the sum of the product {Σ kSAO-i [AO-i]/105} of the kSAO-i and the [AO-i]/105 of AO-i (Tocs and Toc-3s) contained in the vegetable oils.

Measurements of Singlet Oxygen-Quenching Activity of Vitamin E Homologs and Palm Oil and Soybean Extracts in a Micellar Solution.

Recently, a new assay method that can quantify the singlet oxygen-absorption capacity (SOAC) of antioxidants (AO) and food extracts in homogeneous organic solvents has been proposed. In the present study, second-order rate constants (kQ ) for the reaction of singlet oxygen (1 O2 ) with vitamin E homologs (α-, ß-, γ-, and δ-tocopherols [Toc] and α-, ß-, γ-, and δ-tocotrienols [Toc-3]) were measured in an aqueous Triton X-100 (5.0 wt%) micellar solution (pH 7.4). Toc-3 showed kQ values larger than those of Toc in a micellar solution, although Toc and Toc-3 showed the same kQ values in a homogeneous solution. Similar measurements were performed for 5 palm oil extracts 1-5 and one soybean extract 6, which contained different concentrations of Toc, Toc-3, and carotenoids. It has been clarified that the 1 O2 -quenching rates (kQ ) (that is, the relative SOAC value) obtained for extracts 3-6 may be explained as the sum of the product ΣkQAO-iAO-i/100 of the rate constant ( kQAO-i ) and the concentration ([AO-i]/100) of AO-i contained. The UV-vis absorption spectra of Toc and Toc-3 were measured in a micellar solution and chloroform. The results obtained demonstrated that the kQ values of AO in homogeneous and heterogeneous solutions vary notably depending on (1) polarity (dielectric constant [ε]) of the reaction field between 1 O2 and AO, (2) the local concentration of AO, and (3) the mobility of AO in solution. The results suggest that the SOAC method is applicable to the measurement of 1 O2 -quenching activity of general food extracts in a heterogeneous micellar solution.

Kinetic Study of the Aroxyl-Radical-Scavenging Activity of Five Fatty Acid Esters and Six Carotenoids in Toluene Solution: Structure-Activity Relationship for the Hydrogen Abstraction Reaction.

A kinetic study of the reaction between an aroxyl radical (ArO•) and fatty acid esters (LHs 1-5, ethyl stearate 1, ethyl oleate 2, ethyl linoleate 3, ethyl linolenate 4, and ethyl arachidonate 5) has been undertaken. The second-order rate constants (ks) for the reaction of ArO• with LHs 1-5 in toluene at 25.0 °C have been determined spectrophotometrically. The ks values obtained increased in the order of LH 1 < 2 < 3 < 4 < 5, that is, with increasing the number of double bonds included in LHs 1-5. The ks value for LH 5 was 2.93 × 10-3 M-1 s-1. From the result, it has been clarified that the reaction of ArO• with LHs 1-5 was explained by an allylic hydrogen abstraction reaction. A similar kinetic study was performed for the reaction of ArO• with six carotenoids (Car-Hs 1-6, astaxanthin 1, ß-carotene 2, lycopene 3, capsanthin 4, zeaxanthin 5, and lutein 6). The ks values obtained increased in the order of Car-H 1 < 2 < 3 < 4 < 5 < 6. The ks value for Car-H 6 was 8.4 × 10-4 M-1 s-1. The ks values obtained for Car-Hs 1-6 are in the same order as that of the values for LHs 1-5. The results of detailed analyses of the ks values for the above reaction indicated that the reaction was also explained by an allylic hydrogen abstraction reaction. Furthermore, the structure-activity relationship for the reaction was discussed by taking the result of density functional theory calculation reported by Martinez and Barbosa into account.

Synthesis and crystal structure of pyrroloquinoline quinol (PQQH2) and pyrroloquinoline quinone (PQQ).

Pyrroloquinoline quinone (PQQ) is a water-soluble quinone compound first identified as a cofactor of alcohol- and glucose-dehydrogenases (ADH and GDH) in bacteria. For example, in the process of ADH reaction, alcohol is oxidized to the corresponding aldehyde, and inversely PQQ is reduced to pyrroloquinoline quinol (PQQH2). PQQ and PQQH2 molecules play an important role as a cofactor in ADH and GDH reactions. However, crystal structure analysis has not been performed for PQQ and PQQH2. In the present study, the synthesis of PQQH2 powder crystals was performed under air, by utilizing vitamin C as a reducing agent. By reacting a trihydrate of disodium salt of PQQ (PQQNa2·3H2O) with excess vitamin C in H2O at 293 and 343 K, yellowish brown and black powder crystals of PQQH2 having different properties were obtained in high yield, respectively. The former was PQQH2 trihydrate (PQQH2·3H2O) and the latter was PQQH2 anhydrate (PQQH2). Furthermore, sodium-free red PQQ powder crystal (a monohydrate of PQQ, PQQ·H2O) was prepared by the reaction of PQQNa2·3H2O with HCl in H2O. Single crystals of PQQH2 and PQQ were prepared from Me2SO/CH3CN mixed solvent, and we have succeeded in the crystal structure analyses of PQQH2 and PQQ for the first time.

Development of a Singlet Oxygen Absorption Capacity (SOAC) Assay Method. Measurements of the SOAC Values for Carotenoids and α-Tocopherol in an Aqueous Triton X-100 Micellar Solution.

Recently, a new assay method for the quantification of the singlet oxygen absorption capacity (SOAC) of antioxidants (AOs) and food extracts in homogeneous organic solvents was proposed. In this study, second-order rate constants (kQ) for the reaction of singlet oxygen (1O2) with eight different carotenoids (Cars) and α-tocopherol (α-Toc) were measured in an aqueous Triton X-100 (5.0 wt %) micellar solution (pH 7.4, 35 °C), which was used as a simple model of biomembranes. The kQ and relative SOAC values were measured using ultraviolet-visible (UV-vis) spectroscopy. The UV-vis absorption spectra of Cars and α-Toc were measured in both a micellar solution and chloroform, to investigate the effect of solvent on the kQ and SOAC values. Furthermore, decay rates (kd) of 1O2 were measured in 0.0, 1.0, 3.0, and 5.0 wt % micellar solutions (pH 7.4), using time-resolved near-infrared fluorescence spectroscopy, to determine the absolute kQ values of the AOs. The results obtained demonstrate that the kQ values of AOs in homogeneous and heterogeneous solutions vary notably depending on (i) the polarity [dielectric constant (ε)] of the reaction field between AOs and 1O2, (ii) the local concentration of AOs, and (iii) the mobility of AOs in solution. In addition, the kQ and relative SOAC values obtained for the Cars in a heterogeneous micellar solution differ remarkably from those in homogeneous organic solvents. Measurements of kQ and SOAC values in a micellar solution may be useful for evaluating the 1O2 quenching activity of AOs in biological systems.

Kinetic Study of Aroxyl-Radical-Scavenging and α-Tocopherol-Regeneration Rates of Five Catecholamines in Solution: Synergistic Effect of α-Tocopherol and Catecholamines.

Detailed kinetic studies have been performed for reactions of aroxyl (ArO(•)) and α-tocopheroxyl (α-Toc(•)) radicals with five catecholamines (CAs) (dopamine (DA), norepinephrine (NE), epinephrine (EN), and 5- and 6-hydroxydopamine (5- and 6-OHDA)) and two catechins (epicatechin (EC) and epigallocatechin gallate (EGCG)) to clarify the free-radical-scavenging activity of CAs. Second-order rate constants (ks and kr) for reactions of ArO(•) and α-Toc(•) radicals with the above antioxidants were measured in 2-propanol/water (5:1, v/v) solution at 25.0 °C, using single- and double-mixing stopped-flow spectrophotometries, respectively. Both the rate constants (ks and kr) increased in the order NE < EN < DA < EC < 5-OHDA < EGCG < 6-OHDA. The ks and kr values of 6-OHDA are large and comparable to the corresponding values of ubiquinol-10 and sodium ascorbate, which show high free-radical-scavenging activity. The ultraviolet-visible absorption of α-Toc(•) (λmax = 428 nm), which was produced by the reaction of α-tocopherol (α-TocH) with ArO(•), disappeared under the coexistence of CAs due to the α-TocH-regeneration reaction. The results suggest that the CAs may contribute to the protection from oxidative damage in nervous systems, by scavenging free radicals (such as lipid peroxyl radical) and regenerating α-TocH from the α-Toc(•) radical.

Development of Singlet Oxygen Absorption Capacity (SOAC) Assay Method Using a Microplate Reader.

Recently, a new assay method that can quantify the singlet oxygen absorption capacity (SOAC) of natural antioxidants and food extracts was developed. The SOAC values were measured in ethanol-chloroform-D2O (50 + 50 + 1, v/v/v) solution at 35°C using a UV-Vis spectrophotometer equipped with a six-channel cell positioner and an electron-temperature control unit. In the present study, measurement of the SOAC values was performed for eight representative carotenoids and three vegetable extracts (tomato, carrot, and red paprika) using a versatile instrument, the microplate reader. A 24-well glass microplate was used for measurements because a plastic microplate, commonly used in the laboratory, dissolves in the ethanol-chloroform-D2O solution. The SOAC values of eight carotenoids and three vegetable extracts measured using a microplate reader were in good agreement with the corresponding values measured using a UV-Vis spectrophotometer, suggesting that the microplate reader is an applicable instrument for the measurement of reliable SOAC values for general antioxidants and food extracts in solution.

Pyrroloquinoline quinone (PQQ) is reduced to pyrroloquinoline quinol (PQQH2) by vitamin C, and PQQH2 produced is recycled to PQQ by air oxidation in buffer solution at pH 7.4.

Measurements of the reaction of sodium salt of pyrroloquinoline quinone (PQQNa2) with vitamin C (Vit C) were performed in phosphate-buffered solution (pH 7.4) at 25 °C under nitrogen atmosphere, using UV-vis spectrophotometry. The absorption spectrum of PQQNa2 decreased in intensity due to the reaction with Vit C and was changed to that of pyrroloquinoline quinol (PQQH2, a reduced form of PQQ). One molecule of PQQ was reduced by two molecules of Vit C producing a molecule of PQQH2 in the buffer solution. PQQH2, thus produced, was recycled to PQQ due to air oxidation. PQQ and Vit C coexist in many biological systems, such as vegetables, fruits, as well as in human tissues. The results obtained suggest that PQQ is reduced by Vit C and functions as an antioxidant in biological systems, because it has been reported that PQQH2 shows very high free-radical scavenging and singlet-oxygen quenching activities in buffer solutions.

Kinetic study of the quenching reaction of singlet oxygen by seven rice bran extracts in ethanol solution. Development of a singlet oxygen absorption capacity (SOAC) assay method.

Measurements of singlet oxygen ((1)O2) quenching rates (kQ (S)) and the relative singlet oxygen absorption capacity (SOAC) values were performed for seven rice bran extracts 1-7, which contained different concentrations of antioxidants (AOs) (such as α-, ß-, γ-, and δ-tocopherols and -tocotrienols, three carotenoids (lutein, ß-carotene, and zeaxanthin), and γ-oryzanol), in ethanol at 35 °C using UV-vis spectrophotometry. The concentrations of four tocopherols and four tocotrienols, three carotenoids, and γ-oryzanol contained in the extracts were determined using HPLC-MS/MS, UV-HPLC, and UV-vis absorption spectroscopy, respectively. Furthermore, comparisons of kQ (S) (Obsd.) values observed for the above extracts 1-7 with the sum of the product {[Formula: see text] [AO-i]} of the [Formula: see text] values obtained for each AO-i and the concentration ([AO-i]) of AO-i contained in extracts 1-7 were performed. From the results, it has been ascertained that the SOAC method is applicable to general food extracts to evaluate their (1)O2-quenching activity.

Development of singlet oxygen absorption capacity (SOAC) assay method. 4. Measurements of the SOAC values for vegetable and fruit extracts.

Measurements of the second-order rate constants and the singlet oxygen absorption capacity (SOAC) values for the reaction of singlet oxygen ((1)O2) with 23 kinds of food extracts were performed in ethanol/chloroform/D2O (50:50:1, v/v/v) solution at 35 °C. It has been clarified that the SOAC method is useful to evaluate the (1)O2-quenching activity (i.e. the SOAC value) of food extracts having two orders of magnitude different rate constants from 3.18 × 10(4) L g(-1) s(-1) for tomato to 1.55 × 10(2) for green melon. Furthermore, comparison of the observed rate constants for the above food extracts with the calculated ones based on the concentrations of seven kinds of carotenoids included in the food extracts and the rate constants reported for each carotenoids was performed, in order to ascertain the validity of the SOAC assay method developed and to clarify the ratio of the contribution of principal carotenoids to the SOAC value.

Kinetic study of the scavenging reaction of the aroxyl radical by seven kinds of rice bran extracts in ethanol solution. Development of an aroxyl radical absorption capacity (ARAC) assay method.

Recently, a new assay method that can quantify the aroxyl radical (ArO•) absorption capacity (ARAC) of antioxidants (AOHs) was proposed. In the present work, the second-order rate constants (ks(Extract)) and ARAC values for the reaction of ArO• with seven kinds of rice bran extracts 1-7, which contain different concentrations of α-, ß-, γ-, and δ-tocopherols and -tocotrienols (α-, ß-, γ-, and δ-Tocs and -Toc-3s) and γ-oryzanol, were measured in ethanol at 25 °C using stopped-flow spectrophotometry. The ks(Extract) value (1.26 × 10(-2) M(-1) s(-1)) of Nipponbare (extract 1) with the highest activity was 1.5 times larger than that (8.29 × 10(-3)) of Milyang-23 (extract 7) with the lowest activity. The concentrations (in mg/100 g) of α-, ß-, γ-, and δ-Tocs and -Toc-3s and γ-oryzanol found in the seven extracts 1-7 were determined using HPLC-MS/MS and UV-vis absorption spectroscopy, respectively. From the results, it has been clarified that the ArO•-scavenging rates (ks(Extract)) (that is, the relative ARAC value) obtained for the seven extracts 1-7 may be approximately explained as the sum of the product {Σ ks(AOH-i) [AOH-i]/10(5)} of the rate constant (ks(AOH-i)) and the concentration ([AOH-i]/10(5)) of AOH-i (Tocs, Toc-3s, and γ-oryzanol) included in rice bran extracts. The contribution of γ-oryzanol to the ks(Extract) value was estimated to be between 3.0-4.7% for each extract. Taken together, these results suggest that the ARAC assay method is applicable to general food extracts.

Kinetic study of the quenching reaction of singlet oxygen by α-, ß-, γ-, δ-tocotrienols, and palm oil and soybean extracts in solution.

Measurements of the singlet oxygen ((1)O2) quenching rates (kQ (S)) and the relative singlet oxygen absorption capacity (SOAC) values were performed for 11 antioxidants (AOs) (eight vitamin E homologues (α-, ß-, γ-, and δ-tocopherols and -tocotrienols (-Tocs and -Toc-3s)), two vitamin E metabolites (α- and γ-carboxyethyl-6-hydroxychroman), and trolox) in ethanol/chloroform/D2O (50:50:1, v/v/v) and ethanol solutions at 35 °C. Similar measurements were performed for five palm oil extracts 1-5 and one soybean extract 6, which included different concentrations of Tocs, Toc-3s, and carotenoids. Furthermore, the concentrations (wt%) of Tocs, Toc-3s, and carotenoids included in extracts 1-6 were determined. From the results, it has been clarified that the (1)O2-quenching rates (kQ (S)) (that is, the relative SOAC value) obtained for extracts 1-6 may be explained as the sum of the product {Σ kQ(AO-i) (S) [AO-i]/100} of the rate constant (kQ(AO-i) (S)) and the concentration ([AO-i]/100) of AO-i (Tocs, Toc-3s, and carotenoid) included.

Finding of synergistic and cancel effects on the aroxyl radical-scavenging rate and suppression of prooxidant effect for coexistence of α-tocopherol with ß-, γ-, and δ-tocopherols (or -tocotrienols).

Measurements of aroxyl radical (ArO•)-scavenging rate constants (k(s)(AOH)) of antioxidants (AOHs) [α-, ß-, γ-, and δ-tocopherols (TocHs) and -tocotrienols (Toc-3Hs)] were performed in ethanol solution via stopped-flow spectrophotometry. k(s)(AOH) values of α-, ß-, γ-, and δ-Toc-3Hs showed good agreement with those of the corresponding α-, ß-, γ-, and δ- TocHs. k(s)(AOH) values were measured not only for each antioxidant but also for mixtures of two antioxidants: (i) α-TocH with ß-, γ-, or δ-TocH and (ii) α-TocH with α-, ß-, γ-, or δ-Toc-3H. A synergistic effect in which the k(s)(AOH) value increases by 12% for γ-TocH (or by 12% for γ-Toc-3H) was observed for solutions including α-TocH and γ-TocH (or γ-Toc-3H). On the other hand, a cancel effect in which the k(s)(AOH) value decreases (a) by 7% for ß-TocH (or 11% for ß-Toc-3H) and (b) by 24% for δ-TocH (or 25% for δ-Toc-3H) was observed for solutions including two kinds of antioxidants. However, only a synergistic effect may function in edible oils, because contents of ß- and δ-TocHs (and ß- and δ-Toc-3Hs) are much less than those of α- and γ-TocHs (and α- and γ-Toc-3Hs) in many edible oils. UV-vis absorption of α-Toc•, which was produced by reaction of α-TocH with ArO•, decreased remarkably for coexistence of α-TocH with ß-, γ-, or δ-TocH (or ß-, γ-, or δ-Toc-3H), indicating that the prooxidant effect of α-Toc• is suppressed by the coexistence of other TocHs and Toc-3Hs.

A kinetic study of the radical-scavenging action of tocotrienols in the membranes of egg yolk phosphatidylcholine vesicles.

Vitamin E is localized in membranes and functions as an efficient inhibitor of lipid peroxidation in biological systems. In this study, we measured the second-order rate constants (ks) for the reaction of tocotrienol homologues (α-, ß-, γ-, and δ-Toc-3Hs) with the aroxyl radical (ArO•) used as a model for lipid peroxyl radicals (LOO•) in the membranes of egg yolk phosphatidylcholine (EYPC) vesicles by stopped-flow spectrophotometry, and compared them to those of tocopherol homologues (α-, ß-, γ-, and δ-TocHs). The relative rate constants of Toc-3H homologues to α-Toc-3H in membranes (α/ß/γ/δ=100/83.7/63.2/20.2) were not much different to those of TocH homologues to α-TocH (α/ß/γ/δ=100/88.4/83.8/17.3). Each ks value of Toc-3H homologues in membranes was 60-80% of that of the corresponding TocH homologues except for the almost identical ks values of δ-homologues, but there was no difference in EtOH solution between each ks value of the corresponding homologues of Toc-3H and TocH. These results indicate that the difference of the alkyl-side chain structure of vitamin E causes a change in the mobility of vitamin E molecules and/or the location of their antioxidant OH-groups in membranes, resulting in lowered radical-trapping rates of Toc-3Hs. By use of the ratio of the kinh value of α-TocH with LOO• (3.20×10(6) M(-1)s(-1)) to the ks value of α-TocH with ArO• (8.05×10(4) M(-1)s(-1)) in chlorobenzene (that is, 39.8), the kinh value for the reaction of α-TocH with LOO• in membrane was estimated to be 1.03×10(5) M(-1)s(-1).

Kinetic study of aroxyl radical scavenging and α-tocopheroxyl regeneration rates of pyrroloquinolinequinol (PQQH2, a reduced form of pyrroloquinolinequinone) in dimethyl sulfoxide solution: finding of synergistic effect on the reaction rate due to the coexistence of α-tocopherol and PQQH2.

Measurements of aroxyl radical (ArO•)-scavenging rate constants (ks AOH) of antioxidants (AOHs: pyrroloquinolinequinol (PQQH2), α-tocopherol (α-TocH), ubiquinol-10 (UQ10H2), epicatechin, epigallocatechin, epigallocatechin gallate, and caffeic acid) were performed in dimethyl sulfoxide (DMSO) solution, using stopped-flow spectrophotometry. The ks AOH values were measured not only for each AOH but also for the mixtures of two AOHs ((i) α-TocH and PQQH2 and (ii) α-TocH and UQ10H2). A notable synergistic effect that the ks AOH values increase 1.72, 2.42, and 2.50 times for α-TocH, PQQH2, and UQ10H2, respectively, was observed for the solutions including two kinds of AOHs. Measurements of the regeneration rates of α-tocopheroxyl radical (α-Toc•) to α-TocH by PQQH2 and UQ10H2 were performed in DMSO, using double-mixing stopped-flow spectrophotometry. Second-order rate constants (kr) obtained for PQQH2 and UQ10H2 were 1.08 × 105 and 3.57 × 104 M−1 s−1, respectively, indicating that the kr value of PQQH2 is 3.0 times larger than that of UQ10H2. It has been clarified that PQQH2 and UQ10H2 having two HO groups within a molecule may rapidly regenerate two molecules of α-Toc• to α-TocH. The result indicates that the prooxidant effect of α-Toc• is suppressed by the coexistence of PQQH2 or UQ10H2.

Development of a new free radical absorption capacity assay method for antioxidants: aroxyl radical absorption capacity (ARAC).

A new free radical absorption capacity assay method is proposed with use of an aroxyl radical (2,6-di-tert-butyl-4-(4'-methoxyphenyl)phenoxyl radical) and stopped-flow spectroscopy and is named the aroxyl radical absorption capacity (ARAC) assay method. The free radical absorption capacity (ARAC value) of each tocopherol was determined through measurement of the radical-scavenging rate constant in ethanol. The ARAC value could also be evaluated through measurement of the half-life of the aroxyl radical during the scavenging reaction. For the estimation of the free radical absorption capacity, the aroxyl radical was more suitable than the DPPH radical, galvinoxyl, and p-nitrophenyl nitronyl nitroxide. The ARAC value in tocopherols showed the same tendency as the free radical absorption capacities reported previously, and the tendency was independent of an oxygen radical participating in the scavenging reaction and of a medium surrounding the tocopherol and oxygen radical. The ARAC value can be directly connected to the free radical-scavenging rate constant, and the ARAC method has the advantage of treating a stable and isolable radical (aroxyl radical) in a user-friendly organic solvent (ethanol). The ARAC method was also successfully applied to a palm oil extract. Accordingly, the ARAC method would be useful in free radical absorption capacity assay of antioxidative reagents and foods.

Aroxyl-radical-scavenging rate increases remarkably under the coexistence of α-tocopherol and ubiquinol-10 (or vitamin C): finding of synergistic effect on the reaction rate.

Measurements of aroxyl radical (ArO(•))-scavenging rate constants (ks(AOH)) of antioxidants (AOHs) (α-tocopherol (α-TocH), ubiquinol-10 (UQ10H2), and sodium ascorbate (Na(+)AsH(-))) were performed in 2-propanol/water (2-PrOH/H2O, 5/1, v/v) solution using stopped-flow spectrophotometry. ks(AOH) values were measured not only for each AOH but also for the mixtures of two AOHs ((i) α-TocH and UQ10H2 and (ii) α-TocH and Na(+)AsH(-)). A notable synergistic effect that the ks(AOH) values increase 1.6, 2.5, and 6.8 times for α-TocH, UQ10H2, and Na(+)AsH(-), respectively, was observed for the solutions including two kinds of AOHs. Furthermore, measurements of the regeneration rates of α-tocopheroxyl radical (α-Toc(•)) to α-TocH by UQ10H2 and Na(+)AsH(-) were performed in 2-PrOH/H2O using double-mixing stopped-flow spectrophotometry. Second-order rate constants (kr) obtained for UQ10H2 and Na(+)AsH(-) were 2.01 × 10(5) and 1.19 × 10(6) M(-1) s(-1), respectively. In fact, UV-vis absorption of α-Toc(•) (λmax = 428 nm), which had been produced by reaction of α-TocH with ArO(•), disappeared under the existence of UQ10H2 or Na(+)AsH(-) due to the above fast regeneration reaction. The result indicates that the prooxidant effect of α-Toc(•) is suppressed by the coexistence of UQ10H2 or Na(+)AsH(-). As α-TocH, UQ10H2, and ascorbate monoanion (AsH(-)) coexist in relatively high concentrations in plasma, blood, and various tissues, the above synergistic effect, that is, the increase of the free-radical-scavenging rate and suppression of the prooxidant reaction, may function in biological systems.