Notable Effects of Metal Salts on UV–Vis Absorption Spectra of α‑, β‑, γ‑, and δ‑Tocopheroxyl Radicals in Acetonitrile Solution. The Complex Formation between Tocopheroxyls and Metal Cations

The measurements of the UV–vis absorption spectra of α-, β-, γ-, and δ-tocopheroxyl (α-, β-, γ-, and δ-Toc^•) radicals were performed by reacting aroxyl (ArO^•) radical with α-, β-, γ-, and δ-tocopherol (α-, β-, γ-, and δ-TocH), respectively, in acetonitrile solution including three kinds of alkali and alkaline earth metal salts (LiClO₄, NaClO₄, and Mg­(ClO₄)₂) (MX or MX₂), using stopped-flow spectrophotometry. The maximum wavelengths (λ_max) of the absorption spectra of the α-, β-, γ-, and δ-Toc^• located at 425–428 nm without metal salts increased with increasing concentrations of metal salts (0–0.500 M) in acetonitrile and approached some constant values, suggesting (Toc^•···M⁺ (or M²⁺)) complex formations. Similarly, the values of the apparent molar extinction coefficient (ε_max) increased drastically with increasing concentrations of metal salts in acetonitrile and approached some constant values. The result suggests that the formations of Toc^• dimers were suppressed by the metal ion complex formations of Toc^• radicals. The stability constants (<i>K</i>) were determined for Li⁺, Na⁺, and Mg²⁺ complexes of α-, β-, γ-, and δ-Toc^•. The <i>K</i> values increased in the order of NaClO₄ < LiClO₄ < Mg­(ClO₄)₂, being independent of the kinds of Toc^• radicals. Furthermore, the <i>K</i> values increased in the order of δ- < γ- < β- < α-Toc^• radicals for each metal salt. The alkali and alkaline earth metal salts having a smaller ionic radius of the cation and a larger charge of the cation gave a larger shift of the λ_max value, a larger ε_max value, and a larger <i>K</i> value. The result of the DFT molecular orbital calculations indicated that the α-, β-, γ-, and δ-Toc^• radicals were stabilized by the (1:1) complex formation with metal cations (Li⁺, Na⁺, and Mg²⁺). Stabilization energy (<i>E</i>_S) due to the complex formation increased in the order of Na⁺ < Li⁺ < Mg²⁺ complexes, being independent of the kinds of Toc^• radicals. The calculated result also indicated that the metal cations coordinate to the O atom at the sixth position of α-, β-, γ-, and δ-Toc^• radicals

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-<i>tert</i>-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 <i>p</i>-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

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 (<i>k</i>_s^Extract) and ARAC values for the reaction of ArO• with seven kinds of rice bran extracts 1–7, which contain different concentrations of α-, β-, γ-, and δ-tocopherols and -tocotrienols (α-, β-, γ-, and δ-Tocs and -Toc-3s) and γ-oryzanol, were measured in ethanol at 25 °C using stopped-flow spectrophotometry. The <i>k</i>_s^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-, β-, 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 (<i>k</i>_s^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 <i>k</i>_s^{AOH‑<i>i</i>} [AOH-<i>i</i>]/10⁵} of the rate constant (<i>k</i>_s^{AOH‑<i>i</i>}) and the concentration ([AOH-<i>i</i>]/10⁵) of AOH-<i>i</i> (Tocs, Toc-3s, and γ-oryzanol) included in rice bran extracts. The contribution of γ-oryzanol to the <i>k</i>_s^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

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 (<i>k</i>_Q) 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 <i>k</i>_Q 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 <i>k</i>_Q and SOAC values. Furthermore, decay rates (<i>k</i>_d) 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 <i>k</i>_Q values of the AOs. The results obtained demonstrate that the <i>k</i>_Q 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 <i>k</i>_Q and relative SOAC values obtained for the Cars in a heterogeneous micellar solution differ remarkably from those in homogeneous organic solvents. Measurements of <i>k</i>_Q and SOAC values in a micellar solution may be useful for evaluating the ¹O₂ quenching activity of AOs in biological systems

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

<div><p>Measurements of the singlet oxygen (¹O₂) quenching rates (<i>k</i>_<i>Q</i> (<i>S</i>)) and the relative singlet oxygen absorption capacity (SOAC) values were performed for 11 antioxidants (AOs) (eight vitamin E homologues (α-, β-, 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 ¹O₂-quenching rates (<i>k</i>_<i>Q</i> (<i>S</i>)) (that is, the relative SOAC value) obtained for extracts 1–6 may be explained as the sum of the product {Σ <i>k</i>_<i>Q</i>^AO-<i>i</i> (<i>S</i>) [AO-<i>i</i>]/100} of the rate constant (<i>k</i>_<i>Q</i>^AO-<i>i</i> (<i>S</i>)) and the concentration ([AO-<i>i</i>]/100) of AO-<i>i</i> (Tocs, Toc-3s, and carotenoid) included.</p></div

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

<p>Measurements of singlet oxygen (¹O₂) quenching rates (<i>k</i>_Q (<i>S</i>)) 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-, β-, γ-, and δ-tocopherols and -tocotrienols, three carotenoids (lutein, β-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 <i>k</i>_Q (<i>S</i>) (Obsd.) values observed for the above extracts 1–7 with the sum of the product { [AO-<i>i</i>]} of the values obtained for each AO-<i>i</i> and the concentration ([AO-<i>i</i>]) of AO-<i>i</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 ¹O₂-quenching activity.</p> <p>Measurements of the quenching rate (<i>k</i>_Q) of singlet oxygen were performed for seven rice bran extracts in ethanol to evaluate their singlet oxygen quenching activity.</p