Finding of Synergistic and Cancel Effects on the Aroxyl Radical

Article pubs.acs.org/JAFC

Finding of Synergistic and Cancel Effects on the Aroxyl RadicalScavenging Rate and Suppression of Prooxidant Effect for Coexistence of α‑Tocopherol with β‑, γ‑, and δ‑Tocopherols (or -Tocotrienols) Aya Ouchi,† Shin-ichi Nagaoka,† Tomomi Suzuki,‡ Katsuhiro Izumisawa,‡ Taisuke Koike,§ and Kazuo Mukai*,† †

Department of Chemistry, Faculty of Science, Ehime University, Matsuyama 790-8577, Japan Eisai Company, Ltd., Koishikawa 5-5-5, Bunkyo-ku, Tokyo 112-8088, Japan § Eisai Food & Chemical Company, Ltd., Nihonbashi 2-13-10, Chuo-ku, Tokyo 103-0027, Japan ‡

S Supporting Information *

ABSTRACT: Measurements of aroxyl radical (ArO•)-scavenging rate constants (ksAOH) of antioxidants (AOHs) [α-, β-, γ-, and δ-tocopherols (TocHs) and -tocotrienols (Toc-3Hs)] were performed in ethanol solution via stopped-flow spectrophotometry. ksAOH values of α-, β-, γ-, and δ-Toc-3Hs showed good agreement with those of the corresponding α-, β-, γ-, and δ- TocHs. ksAOH values were measured not only for each antioxidant but also for mixtures of two antioxidants: (i) α-TocH with β-, γ-, or δ-TocH and (ii) α-TocH with α-, β-, γ-, or δ-Toc-3H. A synergistic effect in which the ksAOH 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 ksAOH value decreases (a) by 7% for β-TocH (or 11% for β-Toc-3H) and (b) by 24% for δ-TocH (or 25% for δ-Toc3H) was observed for solutions including two kinds of antioxidants. However, only a synergistic effect may function in edible oils, because contents of β- and δ-TocHs (and β- and δ-Toc-3Hs) are much less than those of α- and γ-TocHs (and α- and γ-Toc3Hs) 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 β-, γ-, or δ-TocH (or β-, γ-, or δ-Toc-3H), indicating that the prooxidant effect of αToc• is suppressed by the coexistence of other TocHs and Toc-3Hs. KEYWORDS: free radicals, vitamin E, tocopherols, tocotrienols, antioxidant activity, reaction rate, stopped-flow spectrophotometry, coexistence of antioixidants, synergistic and cancel effects

■

where UQ10H• denotes the ubisemiquinone radical. The results of kinetic studies for reactions 3 and 4 indicated that both reactions are important for the antioxidant actions of UQ10H2.11−15 On the other hand, vitamin C (ascorbate monoanion, AsH−) is a representative water-soluble antioxidant. Hydrophilic AsH− also enhances the antioxidant activity of α-TocH by regenerating α-Toc• to α-TocH (reaction 5):15−18

INTRODUCTION α-Tocopherol (α-TocH) is well-known as one of the most important lipophilic antioxidants (AOHs) in foods and biological systems.1−3 The antioxidant action of α-TocH has been ascribed to the scavenging reaction of lipid peroxyl (LOO•) radical, producing the corresponding α-tocopheroxyl (α-Toc•) radical (reaction 1).1 On the other hand, if α-TocH exists in edible oils and biomembranes, α-Toc• radicals may react with unsaturated lipids (LHs) (reaction 2). Reaction 2 is known as a prooxidant reaction, which induces degradation of unsaturated lipids.4−8 k inh

•

LOO + α‐TocH ⎯→ ⎯ LOOH + α‐Toc

•

kp

α‐Toc• + LH → α‐TocH + L•

kr

α‐Toc• + AsH− → α‐TocH + As− •

where As− • is an ascorbate free radical. As described above, αToc• is an important key radical, which appears in the process of antioxidant and prooxidant actions of α-TocH (see reactions 1, 2, 4, and 5). In previous works, we measured the second-order reaction rates (ksAOH) of many kinds of natural antioxidants, such as α-, β-, γ-, and δ-TocHs,19,20 biological hydroquinones including UQ10H2,20 vitamin C (or sodium ascorbate),21 and many polyphenols including catechins and flavone derivatives,21,22

(1) (2)

Ubiquinol-10 (UQ10H2) is also well-known as a representative lipophilic antioxidant.9,10 UQ10H2 functions as an antioxidant by (i) scavenging LOO• (reaction 3) and (ii) regenerating α-Toc• to α-TocH (reaction 4):1,9,10 k inh

LOO• + UQ 10H 2 ⎯→ ⎯ LOOH + UQ 10H• kr

α‐Toc• + UQ 10H 2 → α‐TocH + UQ 10H• © 2014 American Chemical Society

(5)

Received: Revised: Accepted: Published:

(3) (4) 8101

April 30, 2014 July 10, 2014 July 22, 2014 July 22, 2014 dx.doi.org/10.1021/jf502049k | J. Agric. Food Chem. 2014, 62, 8101−8113

Journal of Agricultural and Food Chemistry

Article

homologues. As described above, ArO•-scavenging rates (ksAOH) of α-, β-, γ-, and δ-tocopherols have been investigated in previous works.19,20 However, a detailed kinetic study has not been performed for α-, β-, γ-, and δ-tocotrienols, as far as we know. In the present work, first, measurements of the rate constants (ksAOH) were performed for eight vitamin E homologues in ethanol solution at 25 °C by stopped-flow spectrophotometry, to investigate the effect of alkyl side chain of α-, β-, γ-, and δ-TocHs and -Toc-3Hs on the scavenging rate. Second, measurements of the ksAOH value were performed for solutions including two kinds of antioxidants, (i) α-TocH with β-, γ-, and δ-TocHs and (ii) αTocH with α-, β-, γ-, and δ-Toc-3Hs, to investigate the synergistic and cancel effects of antioxidants on the ArO•scavenging rate. Third, the effect of coexistence of α-TocH and other TocHs (or Toc-3Hs) on the prooxidant action of α-TocH has been studied. Vitamin E homologues are included in many kinds of edible oils such as rapeseed, soybean, peanut, rice bran, palm oils, etc.29−33 Consequently, it will be of interest to study the effect of coexistence of α-TocH with other TocHs (or Toc3Hs) on the antioxidant and prooxidant actions of α-TocH.

with 2,6-di-t-butyl-4-(4-methoxyphenyl)phenoxyl (aroxyl, ArO•) (see Figure 1) in ethanol (reaction 6), by use of

■

Materials. D-α-, β-, γ-, and δ-Tocopherols and -tocotrienols were kindly supplied from Eisai Food & Chemical, Co. Ltd., Japan. The purities of tocopherols and tocotrienols used were as follows; α-, β-, γ-, and δ-TocHs (99.9%, 99.8%, 100.0%, and 99.5%) and α-, β-, γ-, and δToc-3Hs (99.4%, 99.4%, 98.7%, and 98.7%), respectively. ArO• radical was prepared according to the method of Rieker and Scheffler.34 Measurements. Measurement of the second-order rate constant (ksAOH) for reaction of ArO• with AOH (reaction 6) was performed on a Unisoku single-mixing stopped-flow spectrophotometer (Model RSP1000) by mixing equal volumes of ethanol solutions of ArO• and AOH under nitrogen atmosphere.20,21 The time between mixing the two solutions and recording the first data point (that is, dead time) was 10− 20 ms. The reaction was monitored with either single-wavelength detection (Figure 2B) or photodiode-array detection (Figure 2A) attached to the stopped-flow spectrophotometer. All measurements were performed at 25.0 ± 0.5 °C. Experimental errors in the rate constants (ksAOH) were estimated to be about 5% in ethanol solution.

Figure 1. Molecular structures of α-, β-, γ-, and δ-tocopherols (TocHs); α-, β-, γ-, and δ-tocotrienols (Toc-3Hs); and aroxyl radical (ArO•).

stopped-flow spectrophotometry. ArO• can be regarded as a model for active oxygen radicals (LOO• and others) in biological systems, as described in previous works.19,20,23 ks AOH

ArO• + AOH ⎯⎯⎯⎯⎯→ ArOH + AO•

MATERIALS AND METHODS

■

(6)

RESULTS Measurements of Aroxyl Radical-Scavenging Rates for α-, β-, γ-, and δ-Tocopherols and -Tocotrienols in Ethanol Solution. Measurement of the rate constant (ksα‑TocH) for reaction of ArO• radical with α-TocH was performed in ethanol solution (reaction 6). Upon reaction of α-TocH with ArO• radical, the absorbance at 376 and 580 nm of ArO• decreases and the absorbance at 428 nm of α-Toc• radical increases, as shown in Figure 2A. The scavenging rate of ArO• was measured by following the decrease in absorbance at 376 or 580 nm of the ArO• radical, as shown in Figure 2B.28 The pseudo-first-order rate constants (kobsd) at 376 or 580 nm were linearly dependent on the concentration of α-TocH [α-TocH], and thus, the rate equation is expressed as

As described above, α-TocH, UQ10H2, and vitamin C are the most important natural antioxidants in biological systems. These antioxidants coexist in relatively high concentrations in human (and rat) plasma and various tissues.24−27 Furthermore, it is wellknown that various antioxidants coexist in many foods.2 However, a detailed kinetic study has not been performed for mixtures of these antioxidants. Therefore, recently, measurements of ArO•-scavenging rate constants (ksAOH) of antioxidants [α-TocH, UQ10H2, and sodium ascorbate (Na+AsH−)] were performed in 2-propanol/ water (5/1 v/v) solution by stopped-flow spectrophotometry. ksAOH values were measured not only for each antioxidant but also for mixtures of two antioxidants: (i) α-TocH with UQ10H2 and (ii) α-TocH with Na+AsH−.28 A notable synergistic effect in which ksAOH values increase 1.6, 2.5, and 6.8 times for α-TocH, UQ10H2, and Na+AsH−, respectively, was observed for solutions including two kinds of antioxidants. Furthermore, formation of α-Toc• radical was suppressed remarkably for coexistence of αTocH with UQ10H2 (or Na+AsH−). The result suggests that the prooxidant effect induced by the reaction between α-Toc• radical and LH (reaction 2) may be suppressed by the coexistence of αTocH with UQ10H2 (or Na+AsH−). α-, β-, γ-, and δ-Tocopherols and -tocotrienols (α-, β-, γ-, and δ-TocH and -Toc-3H; see Figure 1) are well-known as vitamin E

−d[ArO• ]/dt = kobsd[ArO• ] = ks α‐TocH[α‐TocH][ArO• ] (7) α‑TocH

where ks is the second-order rate constant for oxidation of α-TocH by ArO• radical. ksα‑TocH was obtained by plotting kobsd against [α-TocH], as shown in Figure 2C. The ksα‑TocH(alone) value obtained is 5.14 × 103 M−1·s−1 (see Table 1), where ksα‑TocH(alone) means the ArO• radical-scavenging rate constant obtained in solution including only one antioxidant (α-TocH). As described above, upon reaction of α-TocH with ArO• in 8102

dx.doi.org/10.1021/jf502049k | J. Agric. Food Chem. 2014, 62, 8101−8113


Article

Figure 2. (A) Change in electronic absorption spectra of ArO• and α-Toc• radicals during reaction of ArO• with α-TocH in ethanol solution at 25.0 °C. Initial concentrations were [ArO•] = 8.78 × 10−5 M and [α-TocH] = 9.53 × 10−4 M. Spectra were recorded at 36 ms intervals. Arrow indicates a decrease (ArO•) and an increase (α-Toc•) in absorbance with time. Also shown is the time dependence of absorbance of (B) ArO• radical (at 376 nm) and (D) αToc• radical (at 428 nm) in solutions including five and four different concentrations of α-TocH at 25.0 °C, respectively. (C) Pseudo-first-order rate constant (kobsd) versus [α-TocH] plot.

ethanol, α-Toc• is produced rapidly. α-Toc• is unstable at 25.0 °C and its absorption peak decreases gradually after passing through the maximum (see Figure 2D).28,35 Similar measurements were performed for reaction of ArO• with β-, γ-, and δ-tocopherols and -tocotrienols in ethanol solution. For example, the result obtained for γ-TocH is shown in Figure 3. Upon reaction of γ-TocH with ArO• radical, the absorbance at 376 nm of ArO• decreases rapidly, and an absorption spectrum due to γ-Toc• radical appears at λmax = 432 nm, as shown in Figure 3A. However, the absorption observed for γ-Toc• is very weak; its absorption peak (at 432 nm) decreases rapidly after passing through the maximum and disappears almost at 10 s, differing from the case of α-Toc• (see Figures 2D and 3D).35 The rate constant, ksγ‑TocH(alone), was obtained by plotting kobsd against [γ-TocH] (see Figure 3C). In the present work, measurements of ksAOH(alone) values were repeated three or four times for each α-, β-, γ-, and δ-TocH and -Toc-3H to obtain reliable ksAOH(alone) values, to compare the values of α-, β-, γ-, and δ-tocotrienols with those of α-, β-, γ-, and δ-tocopherols. Each measured ksAOH(alone) value and the average ksAOH(alone) values obtained are summarized in Table 1. As is clear from the ksAOH(alone) values listed in Table 1, the

ArO•-scavenging activity in ethanol increases in the order shown in eq 8: δ‐TocH ∼ δ‐Toc‐3H < γ‐TocH ∼ γ‐Toc‐3H ∼ β‐TocH ∼ β‐Toc‐3H < α‐TocH ∼ α‐Toc‐3H

(8)

Measurements of Aroxyl Radical-Scavenging Rates (ksAOH) for Mixtures of α-Tocopherol with β-, γ-, or δTocopherol in Ethanol. Measurements of ksβ,γ,δ‑TocH(+αTocH) values were performed for solutions including α-TocH and β-, γ-, or δ-TocH, respectively. The notation ksβ,γ,δ‑TocH(+αTocH) indicates the rate constant obtained in solution including two tocopherol components. First, measurement of ksγ‑TocH(+αTocH) was performed by keeping [α-TocH] constant at 2.88 × 10−4 M and varying [γ-TocH] from 0 to 1.21 × 10−3 M (denoted measurement 1 in Table 2). Upon mixing the solution of ArO• radical with the solution including α-TocH and γ-TocH, absorption of the ArO• at 376 and 580 nm decreases rapidly, as shown in Figure 4. Absorption due to α-Toc• and γ-Toc• was observed at 428 nm, but absorption intensity decreased with increasing [γ-TocH]. By 8103



Article

Table 1. Second-Order Rate Constants for Reactions of Antioxidants with ArO• Radical, Average Values, and Relative Rate Constants in Ethanol Solution at 25.0 °C antioxidant

meas ksAOH(alone), M−1·s−1

α-TocH α-Toc-3H

β-TocH

β-Toc-3H γ-TocH γ-Toc-3H

δ-TocH

δ-Toc-3H a

avg ksAOH(alone), M−1·s−1

ksAOH(alone)/ksα‑TocH(alone)

3

avg 5.12 × 10 avg 4.97 × 103

1.00 avg 0.971

avg 2.77 × 103

avg 0.541

avg 3.04 × 104

avg 0.594

avg 2.82 × 103

avg 0.551

avg 2.76 × 103

avg 0.539

avg 1.02 × 103

avg 0.199

avg 1.09 × 103

avg 0.213

5.12 × 10 4.76 × 103 4.97 × 103 5.19 × 103 2.74 × 103 2.70 × 103 2.86 × 103 3.11 × 103 2.96 × 103 2.85 × 103 2.78 × 103 2.89 × 103 2.64 × 103 2.76 × 103 1.00 × 103 1.02 × 103 1.02 × 103 1.02 × 103 1.08 × 103 1.09 × 103

3a

Measurement of ksα‑TocH(alone) for α-TocH was performed many times in ethanol. Average value is 5.12 × 103 M−1·s−1.

analyzing the decay curves of ArO• at 376 nm, kobsd values were determined. Figure 5B (●) shows the kobsd versus [γ-TocH] plot. If α-TocH and γ-TocH coexist in solution, reactions 9 and 10 will occur competitively in solution, because the difference in ksα‑TocH(alone) and ksγ‑TocH(alone) values is not too large, as listed in Table 1.

As expected from the gradient in Figure 5B, the ksγ‑TocH(+αTocH) value (3.25 × 103 M−1·s−1) is 1.15 times larger than ksγ‑TocH(alone) (average 2.82 × 103 M−1·s−1) obtained for the solution including only γ-TocH. A synergistic effect due to the coexistence of α-TocH and γ-TocH in solution was observed for the rate constant ksγ‑TocH(+α-TocH). Measurement was repeated twice to ascertain the result obtained above (see measurement 2 in Table 2). The ksγ‑TocH(+α-TocH) value (3.08 × 103 M−1·s−1) obtained showed good agreement with that (3.25 × 103 M−1·s−1) for measurement 1, indicating a similar synergistic effect. As an effect of the coexistence of α-TocH and γ-TocH was observed for the rate constants ksγ‑TocH(+α-TocH), similar measurements were performed for the solutions including αTocH and β-TocH (or δ-TocH), by keeping [α-TocH] constant and varying [β-TocH] (or [δ-TocH]) (see measurements 1 and 2 in Table 2), respectively. Figure 5A,C shows the plots for kobsd versus [β-TocH] and [δ-TocH], respectively. As we may see clearly from the above plots (●), both the rate constantsksβ‑TocH(+α-TocH) and ksδ‑TocH(+α-TocH) decrease by 11% and 24% compared with the corresponding values of ksβ‑TocH(alone) and ksδ‑TocH(alone). Measurements were repeated twice to ascertain the results obtained above (see measurements 1 and 2 in Table 2). The average rate constants are listed in Table 2, indicating a cancel effect due to the coexistence of α-TocH and β-TocH (or δ-TocH). As observed for the reaction of the solution of ArO• with the solution including α-TocH and γ-TocH (see Figure 4A−C), the absorption due to α-Toc• and β-Toc• (or δ-Toc•) at 428 nm decreased with increasing concentration of β-TocH (or δTocH), as shown in Figure 6A,C. The reason will be discussed in the Discussion section. Measurements of Aroxyl Radical-Scavenging Rates (ksAOH) for Mixtures of α-Tocopherol with α-, β-, γ-, or δTocotrienol in Ethanol. As a clear effect of the coexistence of α-TocH and β-, γ-, or δ-TocH was observed for rate constants ksβ,γ,δ‑TocH(+α-TocH), similar measurements were performed for solutions including α-TocH and α-, β-, γ-, or δ-Toc-3H by

ArO• + α‐TocH + γ‐TocH ks α‐TocH

⎯⎯⎯⎯⎯⎯→ ArOH + α‐Toc• + γ‐TocH

(9)

ArO• + α‐TocH + γ‐TocH ks γ‐TocH

⎯⎯⎯⎯⎯⎯→ ArOH + α‐TocH + γ‐Toc•

(10)

In such a case, we can expect that the kobsd value depends on eq 11, if the interaction among α-TocH, γ-TocH, and their radicals is negligible. kobsd = ks α‐TocH(alone)[α‐TocH] + ks γ‐TocH(alone) [γ‐TocH]

(11)

Next we will assume that a synergistic effect is present. By substituting ksα‑TocH(alone) and ksγ‑TocH(alone) values and the value of [α-TocH] (2.88 × 10−4 M) used for measurement 1 (see Table 2) into eq 11, kobsd was plotted against [γ-TocH] (see dotted line in Figure 5B). Furthermore, the result of the kobsd versus [γ-TocH] plot (○) obtained for the solution including only γ-TocH (see eq 7) is also shown in Figure 5B. As described above, measurement 1 was performed by keeping α-TocH at a constant concentration and varying [γ-TocH]. Consequently, the ksγ‑TocH(+α-TocH) value was determined from the gradient of the kobsd versus [γ-TocH] plot (●) in Figure 5B, by use of eq 12: kobsd = ks α‐TocH(alone)[α‐TocH] + ks γ‐TocH( +α‐TocH) [γ‐TocH]

(12) 8104



Article

Figure 3. (A) Change in electronic absorption spectra of ArO• and γ-Toc• radicals during reaction of ArO• with γ-TocH in ethanol solution at 25.0 °C. Initial concentrations were [ArO•] = 7.92 × 10−5 M and [γ-TocH] = 1.63 × 10−3 M. Spectra were recorded at 100 ms intervals. Arrow indicates a decrease (ArO•) and an increase (γ-Toc•) in absorbance with time. Also shown is time dependence of the absorbance of (B) ArO• radical (at 376 nm) and (D) γ-Toc• radical (at 432 nm) in solutions including six and five different concentrations of γ-TocH at 25.0 °C, respectively. (C) Plot of kobsd versus [γ-TocH].

Table 2. Second-Order Rate Constants Obtained for Mixtures of α-Tocopherol with Different Concentrations of β-, γ-, or δTocopherol

a

measurementa

[α-TocH], M

1 2 avg

2.71 × 10−4 2.95 × 10−4

1 2 avg

2.88 × 10−4 2.99 × 10−4

1 2 avg

2.63 × 10−4 3.01 × 10−4

ksTocH(+α-TocH), M−1·s−1

[TocH], M

Mixture of α-TocH + β-TocH (0−1.04) × 10−3 2.47 × 103 (0−1.17) × 10−3 2.65 × 103 2.56 × 103 Mixture of α-TocH + γ-TocH (0−1.21) × 10−3 3.25 × 103 (0−1.31) × 10−3 3.08 × 103 3.17 × 103 Mixture of α-TocH + δ-TocH (0−2.16) × 10−3 7.43 × 102 (0−2.52) × 10−3 8.02 × 102 7.73 × 103

ratio ksTocH(+α-TocH)/ksTocH(alone) 0.892 0.967 0.930 1.15 1.09 1.12 0.728 0.786 0.757

Measurements were performed by keeping [α-TocH] constant and varying [TocH] and were repeated twice to ascertain the results.

ksα,β,γ,δ‑Toc‑3H(+α-TocH) values and ratios of the rate constants ksα,β,γ,δ‑Toc‑3H(+α-TocH)/ksα,β,γ,δ‑Toc‑3H(alone) obtained are listed in Table 3. To investigate the effect of concentration of α-, β-, γ-, and δToc-3Hs on the reaction rates ksα,β,γ,δ‑Toc‑3H(+α-TocH), similar

keeping [α-TocH] constant and varying the concentration of α-, β-, γ-, or δ-Toc-3H. The kobsd versus [Toc-3H] plots (●) for α-, β-, γ-, and δ-Toc-3H, respectively, are shown in Figure S1A−D, Supporting Information. Analysis of the rate constant ksα,β,γ,δ‑Toc‑3H(+α-TocH) was performed by use of eq 12. The 8105



Article

Figure 4. Change in electronic absorption spectra of ArO• and α-Toc• (and γ-Toc•) radicals during reaction of ArO• with a mixture of α-TocH and γTocH in ethanol at 25.0 °C. [ArO•] = 7.92 × 10−5 and [α-TocH] = 2.99 × 10−4 M. Spectra were recorded at 140 or 100 ms intervals. Absorption of αToc• (and γ-Toc•) decreased with increasing concentrations of γ-TocH. [γ-TocH] = (A) 0 M, (B) 3.26 × 10−4 M, (C) 6.53 × 10−4 M, and (D) 1.31 × 10−3 M.

and a cancel effect was observed for β- and δ-Toc-3H, effects of the coexistence were smaller: 7% increase for γ-Toc-3H, 6% decrease for β-Toc-3H, and 7% decrease for δ-Toc-3H. Effect of the coexistence of α-TocH and α-Toc-3H was small and negligible. UV−Visible Absorption Spectra of α-, β-, γ-, and δTocopheroxyl and -Tocotrienoxyl Radicals in Ethanol Solution. Measurements of wavelengths of absorption maxima (λmax) and molar extinction coefficients (εmax) were performed for α-, β-, γ-, and δ-Toc• radicals in ethanol in a previous work.35 By reacting ArO• with α-TocH in ethanol, absorption of α-Toc• is observed, as shown in Figure 2A. λmaxi (i = 1−4) values of αToc• are easily determined from absorption peaks of the spectrum. The example for γ-Toc• is also shown in Figure 3A. As the stability of γ-Toc• is low compared to that of α-Toc•, weak absorption was observed. Similarly, λmaxi values for α-, β-, and γToc-3• were determined. However, as δ-Toc-3• is unstable, we could not observe absorption, even if we use high concentration of δ-Toc-3H. The results obtained are summarized in Table 5. As described above, upon reaction of α-TocH with ArO• in ethanol, α-Toc• is produced rapidly. α-Toc• is unstable at 25.0 °C; its absorption peak decreases gradually after passing through

measurements were repeated for the mixture including different concentrations of α-, β-, γ-, and δ-Toc-3H. ksα,β,γ,δ‑Toc‑3H(+αTocH) values for measurements 1, 2, and 3 and average values are also summarized in Table 3. As listed in Table 3, ksγ‑Toc‑3H(+αTocH) values obtained for measurements 1, 2, and 3 showed good accordance with each other. Similar results were obtained for α-, β-, and δ-Toc-3Hs. As listed in Table 3, a synergistic effect was observed for γ-Toc3H, and a cancel effect was observed for β- and δ-Toc-3H. Furthermore, the degree of increase (average +12%) of the rate constant ksγ‑Toc‑3H(+α-TocH) is similar to that (average +12%) of ksγ‑TocH(+α-TocH). The degrees of decrease (average −13% and −25%) of ksβ‑Toc‑3H(+α-TocH) and ksδ‑Toc‑3H(+α-TocH) are also similar to those (average −7% and −24%) of ksβ‑TocH(+α-TocH) and ksδ‑TocH(+α-TocH), respectively. Similar measurements were performed for solutions including α-TocH and α-, β-, γ-, or δ-Toc-3H by keeping the concentration of α-, β-, γ-, or δ-Toc-3H constant and varying [α-TocH] (see Table 4). The kobsd versus [α-TocH] plots (for constant concentrations of α-, β-, γ-, or δ-Toc-3H) are shown in Figure S2A−D in Supporting Information. The rate constants ksα‑TocH(+α-, β-, γ-, and δ-Toc-3H) obtained are summarized in Table 4. Although a synergistic effect was observed for γ-Toc-3H 8106



Article

Toc• is very small and negligible. Therefore, we can estimate the ε1 value of α-Toc• radical at λmax1 = 428 nm by use of the relationship Abs(α-Toc• at tmax) = ε1[ArO•]t=0, as reported in a previous work.35 The ε1 value of α-Toc• radical obtained was 4370 M−1·cm−1 in ethanol solution. Similarly, upon reaction of γ-TocH with ArO• in ethanol, γToc• is produced, and its absorption peak decreases rapidly after passing through the maximum (see Figure 3D). As shown in Figure 3D, the maximum absorbance at 432 nm of γ-Toc• observed at tmax increases with increasing [γ-TocH] and approaches a constant value. Therefore, we can tentatively estimate the ε1 value of γ-Toc• radical at λmax1 = 432 nm, by use of the relationship Abs(γ-Toc• at tmax) = ε1[ArO•]t=0.35 The ε1 value of γ-Toc• radical obtained was 820 M−1·cm−1 in ethanol solution. Similarly, the ε1 values of β- and δ-Toc• and α-, β-, and γ-Toc-3• radicals were determined (see Table 5). The λmax1 and ε1 values for α-, β-, and γ-Toc-3• show good agreement with those of the corresponding α-, β-, and γ-Toc• in ethanol. The λmax1 value increases in the order α- < β- ≤ γ- < δToc• (and α- < β- ≤ γ-Toc-3•), although the difference in λmax1 value among α-, β-, γ-, and δ-Toc• (and -Toc-3•) is comparatively small. On the other hand, the ε1 value decreases remarkably in the order α- > β- > γ- > δ-Toc• (and α- > β- > γ- > δ-Toc-3•) in ethanol. The reason will be discussed in the Discussion section. Decrease in UV−Visible Absorption of α-Tocopheroxyl Radical for Coexistence of α-Tocopherol with β-, γ-, or δTocopherol (or -Tocotrienol). Upon reaction of ArO• radical with α-TocH, absorption of α-Toc• radical at λmax1 (= 428 nm) appears rapidly and decreases gradually, as shown in Figure 2D. The concentration of α-Toc• radical produced by reaction with ArO• ([ArO•] = 8.78 × 10−5 M) is similar to that of ArO• if a high concentration of α-TocH is used for the reaction, as described in a previous section.35 On the other hand, if γ-TocH coexists in the above solution, absorption of α-Toc• radical (for [γ-TocH] = 0 M) decreases with increasing [γ-TocH], as shown in Figure 4. The time dependence of absorption observed at 428 nm is shown in Figure 6C, where absorption at 428 nm is due to the sum of α-Toc• and γ-Toc•, although the contribution of the latter is much smaller than that of the former (see Figures 2A and 3A and Table 5). The contribution of γ-Toc• is almost negligible at 10 s, as shown in Figure 3D. Consequently, [α-Toc•] at 10 s was calculated from absorbance of α-Toc•, by use of the relationship Abs = ε1[αToc•], where ε1 = 4370 M−1·cm−1). Figure 6D shows the plot of [α-Toc•], at 10 s (●) in Figure 6C, versus [γ-TocH]. The [αToc•] decreases remarkably with increasing [γ-TocH], suggesting that the prooxidant effect induced by α-Toc• is suppressed by the coexistence of γ-TocH. The reason for the decrease of [αToc•] due to the coexistence of γ-TocH will be discussed in the Discussion section. Similar measurements were performed for solutions including α-TocH and β-TocH (or δ-TocH). The time dependence of absorption observed at 428 nm for the coexistence of (i) α-TocH and β-TocH and (ii) α-TocH and δ-TocH is shown in Figure 6A,E, indicating that absorption of Toc• radicals at 428 nm decreases with increasing [β-TocH] or [δ-TocH]. In the case of δ-TocH, δ-Toc• is very unstable, and the contribution of δ-Toc• to absorption is almost negligible at 10 s. The plot of [α-Toc•], at 10 s in Figure 6E, versus [δ-TocH] is shown in Figure 6F. On the other hand, β-Toc• is not so unstable, and its absorption overlaps with that of α-Toc• at the first stage of reactions 9 and 10. The decrease of [α-Toc•] (at 10 s) due to the coexistence of β-, γ-, or δ-TocHthat is, the ratio of [α-Toc•] with β-, γ-, or δ-

Figure 5. Plots of kobsd versus [AOH] for reactions of ArO• radical with (i) AOH only (○) and (ii) mixture of α-TocH and AOH (●). AOH = (A) β-TocH (measurement 1), (B) γ-TocH (measurement 1), and (C) δ-TocH (measurement 2) (see Table 2 and text). Dotted lines show the plots for which any synergistic effect is absent between α-TocH and β-, γ-, or δ-TocHs.

the maximum and disappears by a bimolecular reaction (reaction 13).15,35 2k d

α‐Toc• + α‐Toc• ⎯→ ⎯ nonradical products (NRP)

(13)

As shown in Figure 2D, the maximum absorbance (that is, the concentration) of α-Toc• observed at tmax increases with increasing [α-TocH] and approaches a constant value, because at high [α-TocH], α-Toc• appears rapidly and the decay of α8107



Article

Figure 6. (Left panels) Time dependence of absorbance of Toc• radicals (at 428 nm) in ethanol solutions including five different concentrations of (A) β-, (C) γ-, and (E) δ-TocH at 25.0 °C. (Right panels) Plots of [α-Toc•], at 10 s in corresponding left panels, versus (B) [β-TocH], (D) [γ-TocH], and (F) [δ-TocH].

TocH to [α-Toc•] without β-, γ-, or δ-TocHsis 0.62 at [βTocH] = 1.04 × 10−3, 0.18 at [γ-TocH] = 1.21 × 10−3, and 0.57 at [δ-TocH] = 2.52 × 10−3 M, which are the maximum concentrations of β-, γ-, and δ-TocHs used for measurements. Similar measurements were performed for solutions including α-TocH and α-, β-, γ-, or δ-Toc-3H. The time dependence of

absorption observed at 428 nm for the coexistence of α-TocH and β-, γ-, or δ-Toc-3H is shown in Figure 7panels A, C, and E, respectively. The decrease of [α-Toc•] (at 10 s)that is, the ratio of [α-Toc•] (with β-, γ-, or δ-Toc-3H) to [α-Toc•] without β-, γ-, or δ-Toc-3Hsis 0.74 at [β-Toc-3H] = 1.28 × 10−4, 0.16 at [γ-Toc-3H] = 1.45 × 10−3, and 0.55 at [δ-Toc-3H] = 2.37 × 8108



Article

Table 3. Second-Order Rate Constants Obtained for Mixtures of α-Tocopherol with Different Concentrations of α-, β-, γ-, or δTocotrienol

a

measurementa

[α-TocH], M

1 2 3 avg

2.82 × 10−4 3.09 × 10−4 2.83 × 10−4

1 2 avg

2.63 × 10−4 3.02 × 10−4

1 2 3 avg

3.00 × 10−4 2.96 × 10−4 3.17 × 10−4

1 2 3 avg

2.37 × 10−4 3.01 × 10−4 2.88 × 10−4

ksToc‑3H(+α-TocH), M−1·s−1

[Toc-3H], M

ratio ksToc‑3H(+α-TocH)/ksToc‑3H(alone)

Mixture of α-TocH + α-Toc-3H (0−2.95) × 10−4 5.32 × 103 (0−9.65) × 10−4 5.21 × 103 −3 (0−1.03) × 10 5.07 × 103 5.20 × 103 Mixture of α-TocH + β-Toc-3H (0−4.32) × 10−4 2.70 × 103 −3 (0−1.28) × 10 2.69 × 103 2.70 × 103 Mixture of α-TocH + γ-Toc-3H (0−4.43) × 10−4 2.98 × 103 −3 (0−1.15) × 10 3.21 × 103 −3 (0−1.45) × 10 3.08 × 103 3.09 × 103 Mixture of α-TocH + δ-Toc-3H (0−8.37) × 10−4 8.22 × 102 −3 (0−2.37) × 10 8.55 × 102 (0−2.39) × 10−3 7.94 × 102 8.24 × 102

1.07 1.05 1.02 1.05 0.868 0.865 0.867 1.08 1.16 1.12 1.12 0.754 0.784 0.728 0.755

Measurements were performed by keeping [α-TocH] constant and varying [TocH] and were repeated two or three times to ascertain the results.

Table 4. Second-Order Rate Constants Obtained for Mixtures of Different Concentrations of α-Tocopherol with α-, β-, γ-, or δTocotrienola tocotrienol

[Toc-3H], M

α-Toc-3H β-Toc-3H γ-Toc-3H δ-Toc-3H a

[α-TocH], M

−4

ksα‑TocH(+Toc-3H), M−1·s−1

ratio ksα‑TocH(+Toc-3H)/ksα‑TocH(alone)

5.27 × 10 4.84 × 103 5.48 × 103 4.74 × 104

1.03 0.945 1.07 0.926

−4

(0−7.51) × 10 (0−7.51) × 10−4 (0−8.32) × 10−4 (0−7.51) × 10−4

2.15 × 10 4.99 × 10−4 5.02 × 10−4 1.10 × 10−3

3

Measurements were performed by keeping the concentration of α-, β-, γ-, or δ-Toc-3H constant and varying [α-TocH].

Table 5. Values of UV−Visible Absorption Maxima and Molar Extinction Coefficients of α-, β-, γ-, and δ-Tocopheroxyls and -Tocotrienoxyls in Ethanol Toc• or Toc-3• •

α-Toc α-Toc-3• β-Toc• β-Toc-3• γ-Toc• γ-Toc-3• δ- Toc• δ- Toc-3• a

λmax1, nm (ε1, M−1·cm−1) a

428 (4370) 428 (4280) 431 (1500)a 430 (1620) 432 (820)a 432 (745) 434 (380)a

λmax2, nm (ε2, M−1·cm−1)

387 sh (1630) 387 sh (2120)

λmax4, nm (ε4, M−1·cm−1) 340 sh (3790) 340 sh (3720)

■

10−3 M. The decrease of [α-Toc•] (+[α-Toc-3•]) due to coexistence of α-TocH and α-Toc-3H was negligible, as expected (data not shown). The degree of decrease of [α-Toc•] (at 10 s) increases in the order shown in eq 14:

DISCUSSION Comparison of Aroxyl Radical-Scavenging Rates (ksAOH) of Tocotrienols with Those of Tocopherols in Ethanol Solution. α-, β-, γ-, and δ-TocHs and -Toc-3Hs are well-known as vitamin E homologues. Toc-3Hs have received much attention in recent years.36 Yoshida et al.37 reported that the reactivities of α-, β-, γ-, and δ-Toc-3Hs toward peroxyl radicals are the same as those of the corresponding α-, β-, γ-, and δ-TocHs in acetonitrile solution. On the other hand, Serbinova et al.38 reported that α-Toc-3H has more potent antioxidant activity than α-TocH for lipid peroxidation in rat liver microsomal membranes. Moreover, some biological functions such as cholesterol-lowering, 39−41 anti-cancer, 42,43 anti-inflammatory,44,45 anti-angiogenic,46,47 and neuroprotection48 effects

α‐Toc‐3H ≪ β‐TocH ∼ β‐Toc‐3H < δ‐TocH ∼ δ‐Toc‐3H < γ‐TocH ∼ γ‐Toc‐3H

b

408 (3010) 407 (3220) 409 (1200)

See ref 35. bShoulder.

λmax3, nm (ε3, M−1·cm−1)

(14)

As described above, [α-Toc•] decreases notably with increasing concentration of β-, γ-, or δ-TocH. This result suggests that the prooxidant effect induced by α-Toc• may be suppressed by the coexistence of β-, γ-, or δ-TocH (or β-, γ-, or δToc-3H). The reason will be discussed in the Discussion section. 8109



Article

Figure 7. (Left panels) Time dependence of absorbance of Toc• radicals (at 428 nm) in ethanol solutions including five (or four) different concentrations of (A) β-, (C) γ-, and (E) δ-Toc-3H at 25.0 °C. (Right panels) Plots of [α-Toc•], at 10 s in corresponding left panels, versus (B) [β-Toc3H], (D) [γ-Toc-3H], and (F) [δ-Toc-3H].

have been reported for Toc-3Hs. These findings suggest that Toc-3Hs have a wide variety of health benefits. In the present work, measurements of ArO•-scavenging rate constants, ksAOH(alone), of antioxidants (α-, β-, γ-, and δ-TocHs and -Toc-3Hs) were performed in ethanol solution via stoppedflow spectrophotometry. As listed in Table 1, the ksAOH(alone) values of α-, β-, γ-, and δ-Toc-3Hs show good agreement with

those of the corresponding TocHs in ethanol, indicating that the difference of the alkyl side chains in tocopherols and tocotrienols does not have a significant effect on the reaction rates. Furthermore, λmax1 and ε1 values of α-, β-, γ-, and δ-Toc-3• radicals also agree well with the corresponding values of α-, β-, γ-, and δ-Toc• radicals in ethanol (see Table 5). The result also indicates that the electronic states of the chroman ring in α-, β-, 8110



Article

γ-, and δ-Toc• and -Toc-3• radicals are similar to one another, being independent of the difference in alkyl side chain. Finding of Synergistic and Cancel Effects on the Aroxyl Radical-Scavenging Rates (ks) for the Coexistence of αTocopherol with β-, γ-, or δ-Tocopherol (or -Tocotrienol.). It is well-known that various antioxidants coexist in many foods and plants2 and biological systems.24−27 However, examples of measurement of free radical-scavenging rates for the coexistence of two antioxidants are very limited.49 Recently, it has been found that the ArO• radical-scavenging rates (ksAOH) increase notably for the coexistence of two antioxidants, (i) α-TocH and vitamin C and (ii) α-TocH and UQ10H2, in 2-propanol/water (5:1 v/v) solution.28 In the present work, measurements of ksAOH for α-, β-, γ-, δTocHs and -Toc-3Hs were performed in ethanol solution. The ksAOH values were measured not only for each antioxidant but also for the mixtures of two antioxidants, (i) α-TocH and β-, γ-, or δ-TocH and (ii) α-TocH and α-, β-, γ-, or δ-Toc-3H. A clear synergistic effect in which ksγ‑TocH(+α-TocH) values increase by 12% was observed for solutions including α-TocH and γ-TocH. On the other hand, a cancel effect in which (i) ksβ‑TocH(+αTocH) and (ii) ksδ‑TocH(+α-TocH) decrease by 7% and 24%, respectively, was observed for the solutions including (i) α-TocH with β-TocH and (ii) α-TocH with δ-TocH. Similar results were observed for the solutions including two kinds of antioxidants (αTocH with β-, γ-, or δ-Toc-3H). It is important to know the concentrations of α-, β-, γ-, and δTocHs and -Toc-3Hs included in foods and plants (especially in edible oils). The contents of α-, β-, γ-, and δ-TocHs in rapeseed, soybean, peanut, pumpkin, sunflower, canola, corn, wheat germ, and almond oils have been determined by an HPLC method, showing that α- and γ-TocHs are mainly included in edible oils.29−31 For example, the contents in edible oils are as follows: (i) in peanut oil, concentrations of α-, β-, γ-, and δ-TocH = 73.2, nd, 312.0, and 13.4 mg/kg, respectively; (ii) in sunflower oil, concentrations are 494.2, nd, 131.0, and 9.2 mg/kg, respectively; and (iii) in rapeseed oil, concentrations are 90.9, nd, 527.6, and 6.1 mg/kg, respectively, as reported by Tuberoso et al.30 Here, nd denotes not detected. Contents of β- and δ-TocHs are much less than those of α- and γ-TocHs. The result suggests that a synergistic effect observed for the reaction rate constant ksγ‑TocH(+α-TocH) for the coexistence of α- and γ-TocHs functions mainly in edible oils, to protect the degradation of edible oils. Furthermore, high concentrations of α- and γ-TocHs and -Toc-3Hs are included in rice bran and palm oil.32,33 Contents of β- and δ-TocHs (and -Toc-3Hs) are much less than those of α- and γ-TocHs (and -TocH-3s), suggesting that a synergistic effect functions in rice bran and palm oils. However, the reason why a synergistic effect functions between α- and γTocHs (or γ-TocH-3s) and a cancel effect functions between (i) α-TocH and β-TocH (or β-Toc-3H) and (ii) α-TocH and δTocH (or δ-Toc-3H) is not clear at present. Scheme for Suppression of Prooxidant Effect of αTocopherol for Coexistence of α-Tocopherol with β-, γ-, or δ-Tocopherol (or -Tocotrienol). As described in the Introduction, α-Toc• is an important key radical, which appears in the process of antioxidant and prooxidant actions of α-TocH. As shown in Figures 4, 6, and 7, we could directly ascertain that the [α-Toc•] produced by the reaction with ArO• radical decreases remarkably with the coexistence of β-, γ-, or δ-TocH (or -Toc-3H), by observing the decrease of UV−vis absorption of α-Toc• radical. The result clearly indicates that the prooxidant

effect of α-Toc• (see reaction 2) may be suppressed by the coexistence of α-TocH and another TocH (or Toc-3H). In previous studies, the scheme of the decay reaction of α-, β-, γ-, and δ- Toc • radicals in organic solvents has been investigated.15,35 In polar ethanol, acetonitrile, and chloroform solvents, α-Toc• decays by a simple bimolecular reaction without dimer formation, suggesting the production of α-TocH and αtocopherol-o-quinonemethide (α-Toc-QM) by disproportionation (reaction 15): 2k d

α‐Toc• + α‐Toc• ⎯→ ⎯ α‐TocH + α‐Toc‐QM

(15)

On the other hand, in nonpolar n-hexane, n-heptane, and diethyl ether solvents, bimolecular radical decay of α-Toc• radical involves dimerization (recombination) and disproportionation (reaction 16):35 dimer (α‐Toc − α‐Toc) 2k −16

⎯⎯⎯⎯⇀ α‐Toc• + α‐Toc•

↽⎯⎯⎯⎯⎯⎯ 2k16

2k d

⎯→ ⎯ α‐TocH + α‐Toc‐QM

(16)

where dimer is quinol ether (see Figure 9d in ref 35), although it is unstable and has not been isolated. k16 and k−16 represent the rate constants for formation and breakdown of dimer, respectively. Dimerization reactions of phenoxyl radicals (including α-Toc•) are fast reactions and are characterized by large rate constants (k ≈ 107−109 M−1·s−1) in nonviscous solvents at room temperature.50 As described in the Results section, the absorbances of α-, β-, γ-, and δ-Toc• radicals produced by reaction with ArO• decrease remarkably in the order α-Toc• > β-Toc• > γ-Toc• > δ-Toc• in ethanol solvent. The smaller absorbance obtained for β-, γ-, and δ-Toc• radicals will be due to the fast formation of dimer (fast equilibrium with the dimer) (reaction 17) in these Toc• radical molecules. The steric repulsion due to two ortho-methyl groups on the α-Toc• radical molecule will hinder the formation of quinol-ether-type dimer in polar ethanol solvent. However, dimer formation will become easier with decreasing number of ortho-methyl groups. dimer (γ‐Toc − γ‐Toc) 2k −17

⎯⎯⎯⎯⇀ γ‐Toc• + γ‐Toc•

↽⎯⎯⎯⎯⎯⎯ 2k17

2k d

⎯→ ⎯ γ‐TocH + γ‐Toc‐QM

(17)

As described above, if α-TocH and γ-TocH coexist in ethanol and react with ArO•, reactions 9 and 10 will occur competitively. The α-Toc• and γ-Toc• radicals produced will disappear following reactions 15 and 17 in ethanol, respectively. In addition to these reactions, α-Toc• radical will react with γ-Toc• and disappear following reactions 18a and 18b: 2k −18

dimer (α‐Toc − γ‐Toc) ⎯⎯⎯⎯⇀ α‐Toc• + γ‐Toc• ↽⎯⎯⎯⎯⎯⎯ 2k18

(18a)

2α‐Toc• + 2γ‐Toc• 2k d

⎯→ ⎯ α‐TocH + γ‐Toc‐QM + γ‐TocH + α‐Toc‐QM (18b)

where 2kd includes three types of bimolecular reaction rates between (i) α-Toc• + α-Toc•, (ii) γ-Toc• + γ-Toc•, and (iii) α8111



Article

Toc• + γ-Toc•, and the reaction produces four kinds of products (α-TocH, α-Toc-QM, γ-TocH, and γ-Toc-QM). Formation of dimer (α-Toc−γ-Toc) will proceed rapidly, because steric hindrance for the reaction between α-Toc• and γ-Toc• is smaller than that between α-Toc• and α-Toc•. As the electron-donating character of α-TocH is larger than that of β-, γ-, and δ-TocHs, we may expect that reaction 19 will proceed if α-TocH coexists with β-, γ-, or δ-Toc• in solution.19 In fact, Niki et al.51 ascertained that α-TocH reacts quite rapidly with β-, γ-, and δ-Toc• radicals to give α-Toc• radical and β-, γ-, and δ-TocHs, respectively, by electron spin resonance (ESR) measurement.

effect of α-Toc• may be suppressed by coexistence with another TocH or Toc-3H. The scheme for the decrease of [α-Toc•] due to coexistence of α-TocH with another TocH (or Toc-3H) was discussed on the basis of the results obtained.

■

ASSOCIATED CONTENT

* Supporting Information S

Two figures showing plots of kobsd vs [AOH] and kobsd vs [αTocH] for reactions of ArO• radical with AOH only and with a mixture of α-TocH and AOH. This material is available free of charge via the Internet at http://pubs.acs.org.

■

k19

α‐TocH + β‐, γ‐, or δ‐Toc• → α‐Toc• + β‐, γ‐, or δ‐TocH

AUTHOR INFORMATION

Corresponding Author

(19)

*Telephone 81-89-927-9588; fax 81-89-927-9590; e-mail [email protected].

•

Consequently, if ArO radical reacts with α-TocH and γ-TocH in a solution, α-Toc• and γ-Toc• are produced, and reactions 15, 17, 18a, 18b, and 19 proceed competitively in the solution. On the other hand, even if ArO• radical reacts with α-TocH and δTocH in a solution, dimer (α-Toc−δ-Toc) formation between αToc• and δ-Toc• (see reactions 18a and 18b) does not occur, because δ-Toc• produced is very unstable and disappears rapidly by forming the dimer δ-Toc−δ-Toc. Consequently, only α-Toc• remains in solution and disappears gradually, as shown in Figure 6E. This will be a reason why larger [α-Toc•] remains in solution at 10 s, compared to the case of coexistence of α-TocH and γTocH. In the case of coexistence of α-TocH and β-TocH, reactions 15, 17, 18a, and 18b will proceed competitively in solution. Furthermore, as β-Toc• is comparatively stable, β-Toc• disappears and α-Toc• is formed by reaction 19, and the α-Toc• produced disappears gradually (see Figure 6A) as observed for the case of coexistence of α-TocH and δ-TocH. Similar results were obtained for solution including α-TocH and β-, γ-, or δ-Toc-3H, as shown in Figure 7, indicating that the difference in alkyl side chain does not have a significant effect on the bimolecular radical decay of Toc• and Toc-3• radical. As described above, α-TocH and the other TocHs and Toc3Hs (especially γ-TocH and γ-Toc-3H) coexist in edible oils.29−33 Consequently, the above synergistic effectthat is, suppression of the prooxidant reaction may function in edible oils. In the present work, a kinetic study of the ArO• radicalscavenging activity of eight vitamin E homologues (α-, β-, γ-, and δ-tocopherols and -tocotrienols) has been performed in ethanol solution, by use of stopped-flow spectrophotometry. The ArO• radical-scavenging rates (ksAOH) of α-, β-, γ-, and δ-Toc-3Hs showed good agreement with those of the corresponding α-, β-, γ-, and δ-TocHs, indicating that the difference of the alkyl side chains in tocopherols and tocotrienols does not have a significant effect on the reaction rates. The ksAOH values were measured not only for each antioxidant but also for mixtures of two antioxidants: (i) α-TocH with β-, γ-, or δ-TocH and (ii) αTocH with α-, β-, γ-, or δ-Toc-3H. Synergistic and cancel effects in which the ksAOH values increase by 12% and decrease by 7% (or 24%) were observed for the solutions including two kinds of antioxidants, (i) α-TocH and γ-TocH and (ii) α-TocH and βTocH (or α-TocH and δ-TocH). As contents of α- and γ-TocHs included in general edible oils are much higher than those of βand δ-TocHs, only a synergistic effect may function in edible oils. Furthermore, UV−vis absorption of α-Toc• (at λmax = 428 nm), which had been produced by reaction of α-TocH with ArO•, decreased remarkably for coexistence of α-TocH with β-, γ-, or δTocH (or -Toc-3H). This result indicates that the prooxidant

Funding

This work was partly supported by a Grant-in-Aid for Challenging Exploratory Research (24658123) from Japan Society for the Promotion of Science. Notes

The authors declare no competing financial interest.

■ ■

ACKNOWLEDGMENTS We are very grateful to Dr. Koichi Abe of Eisai Food & Chemical Co., Ltd. for helpful discussion. REFERENCES

(1) Niki, E. Assessment of antioxidant capacity in vitro and in vivo. Free Radical Biol. Med. 2010, 49, 503−515. (2) Finley, J. W.; Kong, A.-N.; Hintze, K. J.; Jeffery, E. H.; Ji, L. L.; Lei, X. G. Antioxidants in foods: State of the science important to the food industry. J. Agric. Food Chem. 2011, 59, 6837−6846. (3) Esterbauer, H.; Dieber-Rotheneder, M.; Striegl, G.; Waeg, G. Role of vitamin E in preventing the oxidation of low-density lipoprotein. Am. J. Clin. Nutr. 1991, 53, 314S−321S. (4) Cillard, J.; Cillard, P.; Cormier, M. Effect of experimental factors on the prooxidant behavior of α-tocopherol. J. Am. Oil Chem. Soc. 1980, 57, 255−261. (5) Terao, J.; Matsushita, S. The peroxidizing effect of α-tocopherol on autoxidation of methyl linoleate in bulk phase. Lipids 1986, 21, 255− 260. (6) Bowry, V. W.; Stocker, R. Tocopherol-mediated peroxidation: The prooxidant effect of vitamin E on the radical-initiated oxidation of human low-density lipoprotein. J. Am. Chem. Soc. 1993, 115, 6029− 6044. (7) Mukai, K.; Noborio, S.; Nagaoka, S. Why is the order reversed ? Peroxyl-scavenging activity and fats-and-oils protecting activity of vitamin E. Int. J. Chem. Kinet. 2005, 37, 605−610. (8) Ouchi, A.; Ishikura, M.; Konishi, K.; Nagaoka, S.; Mukai, K. Kinetic study of the prooxidant effect of α-tocopherol: Hydrogen abstraction from lipids by α-tocopheroxyl radical. Lipids 2009, 44, 935−943. (9) Ernster, L.; Dallner, G. Biochemical, physiological and medical aspects of ubiquinone function. Biochim. Biophys. Acta 1995, 1271, 195− 204. (10) Coenzyme Q: Molecular Mechanisms in Health and Disease; Kagan, V. E.; , Quinn, P. J., Eds.; CRC Press: Boca Raton, FL, 2001. (11) Naumov, V. V.; Khrapova, N. G. Study of the interaction of ubiquinone and ubiquinol with peroxide radicals by the chemiluminescent method. Biophysics (Engl. Transl. Biofizika) 1983, 28, 774−780. (12) Mukai, K.; Kikuchi, S.; Urano, S. Stopped-flow kinetic study of the regeneration reaction of tocopheroxyl radical by reduced ubiquinone-10 in solution. Biochim. Biophys. Acta 1990, 1035, 77−82. (13) Barclay, L. R. C.; Vinqvist, M. R.; Mukai, K.; Itoh, S.; Morimoto, H. Chain-breaking phenolic antioxidants: Steric and electronic effects in 8112



Article

polyalkylchromanols, tocopherol analogs, hydroquinones, and superior antioxidants of the polyalkylbenzochromanol and naphthofuran class. J. Org. Chem. 1993, 58, 7416−7420. (14) Ouchi, A.; Nagaoka, S.; Mukai, K. Tunneling effect in regeneration reaction of vitamin E by ubiquinol. J. Phys. Chem. B 2010, 114, 6601−6607. (15) Gregor, W.; Grabner, G.; Adelwöhrer, C.; Rosenau, T.; Gille, L. Antioxidant properties of natural and synthetic chromanol derivatives: Study by fast kinetics and electron spin resonance spectroscopy. J. Org. Chem. 2005, 70, 3472−3483. (16) Packer, J. E.; Slater, T. F.; Willson, R. L. Direct observation of a free radical interaction between vitamin E and vitamin C. Nature 1979, 278, 737−738. (17) Mukai, K.; Nishimura, M.; Kikuchi, S. Stopped-flow investigation of the reaction of vitamin C with tocopheroxyl radical in aqueous Triton X-100 micellar solutions. J. Biol. Chem. 1991, 266, 274−278. (18) Bisby, R. H.; Parker, A. W. Reaction of ascorbate with the αtocopheroxyl radical in micellar and bilayer membrane systems. Arch. Biochem. Biophys. 1995, 317, 170−178. (19) Nagaoka, S.; Kuranaka, A.; Tsuboi, H.; Nagashima, U.; Mukai, K. Mechanism of antioxidant reaction of vitamin E: Charge transfer and tunneling effect in proton-transfer reaction. J. Phys. Chem. 1992, 96, 2754−2761. (20) Mukai, K.; Tokunaga, A.; Itoh, S.; Kanesaki, Y.; Ohara, K.; Nagaoka, S.; Abe, K. Strucure-activity relationship of the free-radicalscavenging reaction by vitamin E (α-, β-, γ-, δ-tocopherols) and ubiquinol-10: pH dependence of the reaction rates. J. Phys. Chem. B 2007, 111, 652−662 and references cited therein.. (21) Mitani, S.; Ouchi, A.; Watanabe, E.; Kanesaki, Y.; Nagaoka, S.; Mukai, K. Stopped-flow kinetic study of the aroxyl radical-scavenging action of catechins and vitamin C in ethanol and micellar solutions. J. Agric. Food Chem. 2008, 56, 4406−4417. (22) Mukai, K.; Oka, W.; Watanabe, K.; Egawa, Y.; Nagaoka, S.; Terao, J. Kinetic study of free-radical-scavenging action of flavonoids in homogeneous and aqueous Triton X-100 micellar solutions. J. Phys. Chem. A 1997, 101, 3746−3753. (23) Traber, M. G.; Atkinson, J. Vitamin E, antioxidant and nothing more. Free Radical Biol. Med. 2007, 43, 4−15. (24) Homma, Y.; Kondo, Y.; Kaneko, M.; Kitamura, T.; Nyou, W. T.; Yanagisawa, M.; Yamamoto, Y.; Kakizoe, T. Promotion of carcinogenesis and oxidative stress by dietary cholesterol in rat prostate. Carcinogenesis 2004, 25, 1011−1014. (25) Lass, A.; Forster, M. J.; Sohal, R. S. Effects of coenzyme Q10 and αtocopherol administration on their tissue levels in the mouse: Elevation of mitochondrial α-tocopherol by coenzyme Q10. Free Radical Biol. Med. 1999, 26, 1375−1382. (26) Podda, M.; Weber, C.; Traber, M. G.; Packer, L. Simultaneous determination of tissue tocopherols, tocotrienols, ubiquinols, and ubiquinones. J. Lipid Res. 1996, 37, 893−901. (27) Colome, C.; Artuch, R.; Vilaseca, M.-A.; Sierra, C.; Brandi, N.; Lambruschini, N.; Cambra, F. J.; Campistol, J. Lipophilic antioxidants in patients with phenylketonuria. Am. J. Clin. Nutr. 2003, 77, 185−188. (28) Mukai, K.; Ouchi, A.; Nakaya, S.; Nagaoka, S. Aroxyl-radicalscavenging rate increases remarkably under the coexistence of αtocopherol and ubiquinol-10 (or vitamin C): Finding of synergistic effect on the reaction rate. J. Phys. Chem. B 2013, 117, 8378−8391. (29) Kajimoto, G.; Yoshida, H.; Shibahara, A. Changes in ratio of αtocopherol to polyunsaturated fatty acid in oil by heating, and αtocopherol contents in tissue of rats fed with thermally oxidized oil. Nippon Eiyo Shokuryo Gakkaishi (J. Jpn. Soc. Nutr. Food Sci.) 1984, 37, 37−43. (30) Tuberoso, C. I. G.; Kowalczyk, A.; Sarritzu, E.; Cabras, P. Determination of antioxidant compounds and antioxidant activity in commercial oilseeds for food use. Food Chem. 2007, 103, 1494−1501. (31) Hu, J.-N.; Zhu, X.-M.; Adhikari, P.; Li, D.; Kim, I.-H.; Lee, K.-T. Determination of tocopherol contents in refined edible oils using an HPLC method. J. Food Sci. Nutr. 2009, 14, 260−264.

(32) Sookwong, P.; Nakagawa, K.; Murata, K.; Kojima, Y.; Miyazawa, T. Quantitation of tocotrienol and tocopherol in various rice brans. J. Agric. Food Chem. 2007, 55, 461−466. (33) Palm Oil Information Series; Malaysian Palm Oil Promotion Council: Selangor, Malaysia, 1995; pp 18−19. (34) Rieker, A.; Scheffler, K. Die beteiligung von phenylresten an der aroxylmesomerie. Liebigs Ann. Chem. 1965, 689, 78−92. (35) Mukai, K.; Ouchi, A.; Mitarai, A.; Ohara, K.; Matsuoka, C. Formation and decay dynamics of vitamin E radical in the antioxidant reaction of vitamin E. Bull. Chem. Soc. Jpn. 2009, 82, 494−503. (36) Niki, E. Tocotrienols: Vitamin E beyond tocopherols, 2nd ed; Tan, B., Watson, R., Preedy, V., Eds.; CRC/AOCS Press: Boca Raton, FL, 2012, and references cited therein. (37) Yoshida, Y.; Niki, E.; Noguchi, N. Comparative study on the action of tocopherols and tocotrienols as antioxidant: Chemical and physical effects. Chem. Phys. Lipids 2003, 123, 63−75. (38) Serbinoba, E.; Kagan, V.; Han, D.; Packer, L. Free radical recycling and intramembrane mobility in the antioxidant properties of αtocopherol and α-tocotrienol. Free Radical Biol. Med. 1991, 10, 263− 275. (39) Parker, R. A.; Pearce, B. C.; Clark, R. W.; Gordon, D. A.; Wright, J. J. K. Tocotrienols regulate cholesterol production in mammalian cells by post-transcriptional suppression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. J. Biol. Chem. 1993, 268, 11230−11238. (40) Black, T. M.; Wang, P.; Maeda, N.; Coleman, R. A. Palm tocotrienols protect ApoE ± mice from diet-induced atheroma formation. J. Nutr. 2000, 130, 2420−2426. (41) Qureshi, A. A.; Sami, S. A.; Salser, W. A.; Khan, F. A. Dosedependent suppression of serum cholesterol by tocotrienol-rich fraction (TRF25) of rice bran in hypercholesterolemic humans. Atherosclerosis 2002, 161, 199−207. (42) Sakai, M.; Okabe, M.; Tachibana, H.; Yamada, K. Apoptosis induction by γ-tocotrienol in human hepatoma Hep3B cells. J. Nutr. Biochem. 2006, 17, 672−676. (43) Eitsuka, T.; Nakagawa, K.; Miyazawa, T. Down-regulation of telomerase activity in DLD-1 human colorectal adenocarcinoma cells by tocotrienol. Biochem. Biophys. Res. Commun. 2006, 348, 170−175. (44) Nesaretnam, K.; Meganathan, P. Tocotrienols: Inflammation and cancer. Ann. N. Y. Acad. Sci. 2011, 1229, 18−22. (45) Shibata, A.; Nakagawa, K.; Kawakami, Y.; Tsuzuki, T.; Miyazawa, T. Suppression of γ-tocotrienol on UVB induced inflammation in HaCaT keratinocytes and HR-1 hairless mice via inflammatory mediators multiple signaling. J. Agric. Food Chem. 2010, 58, 7013−7020. (46) Nakagawa, K.; Shibata, A.; Yamashita, S.; Tsuzuki, T.; Kariya, J.; Oikawa, S.; Miyazawa, T. In vivo angiogenesis is suppressed by unsaturated vitamin E, tocotrienol. J. Nutr. 2007, 137, 1938−1943. (47) Shibata, A.; Nakagawa, K.; Sookwong, P.; Tsuduki, T.; Tomita, S.; Shirakawa, H.; Komai, M.; Miyazawa, T. Tocotrienol inhibits secretion of angiogenic factors from human colorectal adenocarcinoma cells by suppressing hypoxia-inducible factor-1α. J. Nutr. 2008, 138, 2136−2142. (48) Khanna, S.; Roy, S.; Slivka, A.; Craft, T. K. S.; Chaki, S.; Rink, C.; Notestine, M. A.; DeVries, A. C.; Parinandi, N. L.; Sen, C. K. Neuroprotective properties of the natural vitamin E α-tocotrienol. Stroke 2005, 36, 2258−2264. (49) Niki, E.; Saito, T.; Kawakami, A.; Kamiya, Y. Inhibition of oxidation of methyl linoleate in solution by vitamin E and vitamin C. J. Biol. Chem. 1984, 259, 4177−4182. (50) Denisov, E. T.; Khudyakov, I. V. Mechanisms of action and reactivities of the free radicals of inhibitors. Chem. Rev. 1987, 87, 1313− 1357. (51) Niki, E.; Tsuchiya, J.; Yoshikawa, Y.; Yamamoto, Y.; Kamiya, Y. Oxidation of lipids. XIII. Antioxidant activities of α-, β-, γ-, and δtocopherols. Bull. Chem. Soc. Jpn. 1986, 59, 497−501.

8113


Finding of Synergistic and Cancel Effects on the Aroxyl Radical

Recommend Documents