Kinetic Study of Aroxyl-Radical-Scavenging and Î±-Tocopherol

Article pubs.acs.org/JPCB

Kinetic Study of Aroxyl-Radical-Scavenging and α‑TocopherolRegeneration Rates of Five Catecholamines in Solution: Synergistic Effect of α‑Tocopherol and Catecholamines Kazuo Mukai,* Kanae Nagai, Yoshifumi Egawa, Aya Ouchi, and Shin-ichi Nagaoka Department of Chemistry, Faculty of Science, Ehime University, Matsuyama 790-8577, Japan ABSTRACT: 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 6hydroxydopamine (5- and 6-OHDA)) and two catechins (epicatechin (EC) and epigallocatechin gallate (EGCG)) to clarify the free-radical-scavenging activity of CAs. Secondorder rate constants (ks and kr) for reactions of ArO• and αToc• radicals with the above antioxidants were measured in 2propanol/water (5:1, v/v) solution at 25.0 °C, using singleand 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-TocHregeneration 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. function as AOH in biological systems.12−14 Furthermore, a synergistic effect of free-radical-scavenging activity was observed for the solutions containing DA (or L-3,4-dihydroxyphenylalanine (L-DOPA)) and the α-TocH model (2,2,5,7,8pentamethyl-6-hydroxychroman, PMHC).16 The solution containing CA and the α-TocH model showed higher activity than the simple sum of the activities of CA and α-TocH model. 5- and 6-Hydroxydopamines (5-OHDA and 6-OHDA) (see Figure 1) are the oxidation products of DA by molecular oxygen, and called OHDA (or oxidopamine).17 6-OHDA is a potent neurotoxin that destroys sympathetic nerves, inducing neurodegenerative diseases.18,19 The main use of 6-OHDA in scientific research is to induce Parkinson’s disease in laboratory animals such as mice, rats, and monkeys, to develop and test new medicines and treatments for Parkinson’s disease.20 5-OHDA is a pyrogallol derivative, and 6-OHDA is a hydroxy derivative of p-hydroquinone having the same amino alkyl side chain as that of DA. Consequently, we may expect higher antioxidant activities from 5- and 6-OHDA than those from CAs (DA, EN, and NE).21,22 However, kinetic studies on the free-radical-scavenging activities of 5- and 6-OHDA have not been performed, as far as we know. DA, EN, and NE are called CAs (or CA neurotransmitters). As the molecular

1. INTRODUCTION The lipid peroxyl radical (LOO•) has attracted much attention as one of the representative reactive oxygen species generated in biological systems. LOO• reacts with many kinds of biological targets including lipids, sterols, proteins, DNA, and RNA.1,2 Reactions with LOO• occur mainly by chemical reaction, inducing the degradation of biological systems. Oxidative damage of these biomolecules accumulates with age and causes some neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis.3,4 It is well known that dopamine (DA), DL-norepinephrine (NE), and epinephrine (EN) (see Figure 1) function as neurotransmitters in mammalian central and peripheral nervous systems (especially in brain).5 These molecules are also called catecholamines (CAs) because they contain a catechol and a side-chain amine group in a molecule. Furthermore, these CAs might also function as an endogenous antioxidant (AOH) in nervous systems. In fact, the antioxidant properties of CAs were ascertained by in vitro experiments on neuronal cell6−9 and peripheral blood cells.10,11 Free-radical-scavenging activities of CA neurotransmitters have been studied by several investigators.8,12−15 Kinetic studies on the reaction of CAs with active free radicals (such as tert-butoxyl, cumyloxyl, cumylperoxyl, and galvinoxyl radicals) were carried out in acetonitrile and/or propionitrile solutions. The results suggest that these CAs © XXXX American Chemical Society

Received: April 28, 2016 Revised: June 23, 2016

A

DOI: 10.1021/acs.jpcb.6b04285 J. Phys. Chem. B XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry B

Table 1. Second-Order Rate Constants (ksAOH) for the Reaction of Aroxyl (ArO•) Radical with Seven Kinds of Antioxidants (AOHs) and Related Compounds and Relative Rate Constants (ksAOH/ksDA) in 2-Propanol/Water (5:1, v/v) Solution at 25.0 °C, and Rate Constants (ksAOH) for the Reaction of Galvinoxyl (Galv•) Radical with Three AOHs in CH3CN antioxidant (AOH) DA DL-NE EN 5-OHDA 6-OHDA EC EGCG α-TocH ubiquinol-10 (UQ10H2) sodium ascorbate (Na+AsH−) a

ksAOH/M−1 s−1 ArO• in 2-propanol/water

ksAOH/ksDA ratio

ksAOH/M−1 s−1 Galv• in CH3CN

× × × × × × × × ×

1.00 0.444 0.507 4.42 98.4 4.28 14.3 176 176

23a 8.1a 9.1a

4.91 2.18 2.49 2.17 4.83 2.10 7.03 8.65 8.63

1.02 × 104b

α ‐Toc• + AOH → α ‐TocH + AO•

Figure 1. Molecular structures of five kinds of CAs (DA, NE, EN, 5OHDA, and 6-OHDA), α-tocopherol (α-TocH), α-tocopheroxyl (αToc•) radical, and aroxyl (ArO•) radical.

(2)

From the results, the structure−activity relationship in freeradical-scavenging reactions of CAs has been discussed. Furthermore, the ultraviolet−visible (UV−vis) absorption spectra of the α-Toc• radical were measured under the coexistence of α-TocH and five CAs, expecting a synergistic effect between these AOHs.

structures of OHDAs (5- and 6-OHDA) show, these molecules are not CAs but CA derivatives (Figure 1). However, in the present work, 5- and 6-OHDA were tentatively included in CAs for simplicity. α-, β-, γ-, δ-TocHs, biological hydroquinones including ubiquinol-10 (UQ10H2), vitamin C (Vit C) (or sodium ascorbate (Na+AsH−)), and many polyphenols including catechins and flavone derivatives are well known as representative natural AOHs. In previous studies,23−25 the second-order rate constants (ks) for the reaction of aroxyl radical (ArO•) (see Figure 1) with the above AOHs were measured in ethanol (and/or micellar solution) (reaction 1), using stopped-flow spectrophotometry. ArO• can be regarded as a model for active oxygen radicals (LOO• and others) in biological systems23,26 ks

208

See ref 13. bSee ref 31. kr

ArO• + AOH → ArOH + AO•

10 10 10 102 103 102 102 103 103b

2. EXPERIMENTAL METHODS 2.1. Materials. α-TocH was kindly supplied from Eisai Co., Ltd., Japan. DA, NE, EN, 5-OHDA, and 6-OHDA hydrochlorides were obtained from Sigma. EC and EGCG were obtained from Funakoshi, Japan. The ArO• radical was prepared according to the method of Rieker et al.32 2.2. Methods. The measurement of the rate constant (ks) for the reaction of ArO• with AOH (reaction 1) was performed with a Unisoku single-mixing stopped-flow spectrophotometer by mixing equal volumes of the 2-PrOH/H2O solutions of ArO• and AOH under a nitrogen atmosphere.23,24 The kr values of AOH (reaction 2) were measured with a Unisoku double-mixing stopped-flow spectrophotometer. The secondorder rate constant (kr) for the reaction of α-Toc• with AOH was determined by analyzing the decay curve of α-Toc• as described later.33 The time between mixing two solutions and recording the first data point (i.e., dead time) was 10−20 ms. All of the measurements were performed at 25.0 ± 0.5 °C. The experimental errors in the rate constants (ks and kr) were estimated to be about 5% in the 2-PrOH/H2O solution. Ethanol was generally used for measurements of the ks and kr values of AOHs in previous studies.21,22,24,25,33 However, the 2PrOH/H2O solution was used in the present study because the solubility of CAs is low in ethanol. The CAs used are stable in 2-PrOH/H2O.

(1)

As it is well known, α-TocH, UQ10H2, and Vit C are the most important natural AOHs in biological systems. These AOHs coexist in relatively high concentrations in human (and rat) plasma and various tissues.27−30 Therefore, in a previous work, a detailed kinetic study has been performed not only for each AOH but also for the mixtures of these AOHs (i.e., αTocH and UQ10H2 (or Vit C)), indicating a notable synergistic effect for the mixtures of AOHs.31 In the present study, first, measurements of the rate constant (ks) were performed for the reaction of ArO• radical with five CAs (DA, EN, NE, 5- and 6-OHDA) (see Table 1) (reaction 1) in 2-propanol/water (2-PrOH/H2O) (5:1, v/v) solution at 25.0 °C, using stopped-flow spectrophotometry. Similar measurements were performed for epicatechin (EC) and epigallocatechin gallate (EGCG) for comparison. Second, the rate constant (kr) for the reactions of α-Toc• radical with five CAs (reaction 2) was measured in 2-PrOH/H2O solution, using double-mixing stopped-flow spectrophotometry

3. RESULTS 3.1. Measurements of the Aroxyl-Radical-Scavenging Rates (ks) for Five CAs and Two Catechins in 2-Propanol/ Water Solution. The measurement of the rate constant (ks) for the reaction of ArO• radical with DA was performed in 2B


Article

The Journal of Physical Chemistry B PrOH/H2O (reaction 1). By reacting the ArO• radical with DA, the absorbance of ArO• at 375 and 578 nm decreased rapidly, as shown in Figure 2a. The scavenging rate of ArO• was measured by following the decrease in the absorbance of the

ArO• radical at 375 nm, as shown in Figure 2b.23−25 The pseudo-first-order rate constants (kobsd) at 375 nm were linearly dependent on the concentration of DA ([DA]); thus, the rate equation is expressed as −d[ArO• ]/dt = kobsd[ArO• ] = ks[DA][ArO•]

(3)

where ks is the second-order rate constant for the oxidation of DA by the ArO• radical. The rate constant (ks) was obtained by plotting kobsd against [DA], as shown in Figure 2c. The ks value obtained was 4.91 × 10 M−1 s−1 (see Table 1). Similar measurements were performed for the four kinds of CAs (NE, EN, 5-OHDA, and 6-OHDA). Furthermore, the ks values were measured for the two kinds of catechins (EC and EGCG), which have catechol and pyrogallol moieties in the molecules, respectively. In a previous study,24 measurements of the rate constants (ks) of EC and EGCG were performed in ethanol. However, in the present work, the ks values of EC and EGCG were measured in 2-PrOH/H2O solution to compare them with those of five CAs. The results obtained are listed in Table 1. The ks values increased in the order shown in eq 4 NE < EN < DA < EC < 5‐OHDA < EGCG (4)

< 6‐OHDA

As listed in Table 1, the ks value of NE is similar to that of EN, as expected. The ks value of DA is ∼2 times larger than that of NE. On the other hand, the ks values of 5-OHDA and 6OHDA are 1 and 2 orders of magnitude larger than those of DA, NE, and EN, respectively, in 2-PrOH/H2O. Especially, 6OHDA showed high ArO•-scavenging activity. As shown in Figure 2a, we could not observe the absorption spectrum of the DA• radical, which was produced by hydrogen abstraction from the HO groups in the DA molecule. Similarly, absorptions of CA• radicals were not observed (data are not shown) because of the instability of CA• radicals, as reported for EC and EGCG radicals.34 3.2. Measurements of the α-TocH-Regeneration Rates (kr) for Five CAs in 2-Propanol/Water Solution. By reacting the ArO• radical with α-TocH, the absorbance of the ArO• decreases at 375 and 578 nm and the absorbance of the α-Toc• radical increases rapidly at 428 nm, as shown in Figure 3a. α-Toc• is unstable at 25.0 °C; its absorption peak decreases gradually after passing through the maximum at ∼1 s and disappears by a bimolecular reaction (reaction 5) (see Figure 3b)35 2k d

α ‐Toc• + α ‐Toc• ⎯→ ⎯ non‐radical products

(5)

As described in Section 2.2, the measurement of the kr values for the reaction of the α-Toc• radical with CAs (reaction 2) was performed in 2-PrOH/H2O solution, using a double-mixing stopped-flow spectrophotometer.33 The α-Toc• radical was prepared by the first mixing of equal volumes of α-TocH (cell A) and ArO• (cell B) solutions (reaction 1), as described above (see Figure 3a,b), and after 2 s, the second mixing of equal volumes of α-Toc• solution and CA (e.g., 6-OHDA) solution (cell C) (reaction 2) was made. The typical concentrations in cells A and B are 3.64 × 10−4 and 5.29 × 10−5 M, respectively. The decay curves of the absorbance of α-Toc• at 428 nm for the reaction of α-Toc• with 6-OHDA in 2-PrOH/H2O are shown in Figure 3c, indicating that the decay rates increase with increasing [6-OHDA].

Figure 2. (a) Change in the electronic absorption spectra of the ArO• radical during the reaction of ArO• with DA in 2-propanol/water (5:1, v/v) solution at 25.0 °C. The initial concentrations are [ArO•] = 5.90 × 10−5 M and [DA] = 2.26 × 10−2 M. Spectra were recorded at 320 ms intervals. (b) Time dependences of the absorbance of ArO• radical at 375 nm in 2-propanol/water including six different concentrations of DA at 25.0 °C. (c) Pseudo-first-order rate constant (kobsd) vs [DA] plot. C


Article


Figure 3. (a) Change in the electronic absorption spectra of ArO• and α-Toc• radicals during the reaction of ArO• with α-TocH in 2-propanol/ water (5:1, v/v) solution at 25.0 °C. The initial concentrations are [ArO•] = 5.29 × 10−5 M and [α-TocH] = 3.64 × 10−4 M. Spectra were recorded at 120 ms intervals. (b) Time dependences of the absorbance of the α-Toc• radical at 428 nm in solutions containing five different concentrations of α-TocH at 25.0 °C. (c) Time dependences of the absorbance of the α-Toc• radical at 428 nm in 2-propanol/water solution containing five different concentrations of 6-OHDA at 25.0 °C. (d) Pseudo-first-order rate constant (kobsd) vs [6-OHDA] plot.

Table 2. Second-Order Rate Constants (krAOH) for the Reaction of α-Tocopheroxyl (α-Toc•) Radical with Seven Kinds of Antioxidants (AOHs) and Related Compounds and Relative Rate Constants (krAOH/krDA) in 2-Propanol/Water (5:1, v/v) Solution at 25.0 °C

The pseudo-first-order rate constants (kobsd) observed at 428 nm were linearly dependent on [6-OHDA]; thus, the rate equation is expressed as follows −d[α ‐Toc•]/dt = kobsd[α ‐Toc•] = k r[6‐OHDA][α ‐Toc•] (6)

antioxidant

The kr value was obtained by plotting kobsd against [6-OHDA], as shown in Figure 3d. Similar measurements were performed for the reaction of αToc• with four CAs (NE, EN, DA, and 5-OHDA) in 2-PrOH/ H2O solution. The kr values obtained are listed in Table 2. In a previous study, measurements of the kr values of EC and EGCG were performed in ethanol/H2O (5:1, v/v) solution (see Table 2).36 These kr values were used for the comparison. The kr values obtained increased in the order shown in eq 7

DA DL-NE

EN 5-OHDA 6-OHDA EC EGCG ubiquinol-10 sodium ascorbate ethyl linoleate

NE < EN < DA < EC < 5‐OHDA < EGCG < 6‐OHDA

krAOH/M−1 s−1

krAOH/krDA ratio

× × × × × × × × × × ×

1.00 0.326 0.715 40.6 864

2.35 7.67 1.68 9.55 2.03 7.58 2.39 2.01 1.19 8.33 2.7

2

10 10 102 103 105 102a 104a 105b 106b 10−2c 10−2d

855 5060

a

See ref 36. Measurement was performed in ethanol/water (5:1, v/v) solution. bSee ref 31. cSee ref 41. Measurement was performed in toluene at 25.0 °C. dSee ref 42. Measurement was performed in ethanol at 37 °C.

(7)

Especially, the kr value (2.03 × 105 M−1 s−1) obtained for 6OHDA is very large, that is, about 3 orders of magnitude larger than that for DA (2.35 × 102 M−1 s−1), NE (7.67 × 10 M−1 s−1), and EN (1.68 × 102 M−1 s−1). D


Article


Figure 4. Change in the electronic absorption spectra of the ArO• and α-Toc• radicals during the reaction of ArO• with a mixture of α-TocH and DA in 2-propanol/water at 25.0 °C. Initial concentrations are [ArO•] = 5.90 × 10−5 M and [α-TocH] = 1.95 × 10−4 M. Spectra were recorded at 120 ms intervals. The maximum absorption of α-Toc• at 428 nm decreased with increasing concentrations of DA. [DA] = (a) 0, (b) 4.51 × 10−3, (c) 9.03 × 10−3, (d) 1.35 × 10−2, and (e) 1.81 × 10−2 M. (f) Time dependences of the absorbance of α-Toc• radical at 428 nm in 2-propanol/water containing five different concentrations of DA at 25.0 °C.

example, the ratio kr6‑OHDA/krDA = 864 is 8.78 times larger than the ratio ks6‑OHDA/ksDA = 98.4. 3.3. Decrease in the UV−Vis Absorption of the αTocopheroxyl Radical under the Coexistence of α-TocH and Five CAs. As described in a previous section, by reacting

By comparing eqs 4 and 7, both the ks and kr values were found to increase in the order NE < EN < DA < EC < 5OHDA < EGCG < 6-OHDA, independent of the kinds of radicals (ArO• and α-Toc•). However, the relative ratios (ksAOH/ksDA and krAOH/krDA) are different from each other. For E


Article

The Journal of Physical Chemistry B the ArO• radical with α-TocH, the absorption of α-Toc• radical increases rapidly and decreases gradually, as shown in Figure 4a,f. On the other hand, for example, when DA coexists in the above solution, the absorption of α-Toc• radical at λmax (= 428 nm) decreases largely with increasing [DA] and almost disappears at high [DA], as shown in Figure 4a−e. The time dependence of the absorbance of α-Toc• observed at 428 nm is shown in Figure 4f, indicating that the absorbance at 428 nm decreases remarkably with increasing [DA]. Similar measurements were performed for solutions containing α-TocH and the other CAs (NE, EN, 5-OHDA, and 6-OHDA). As observed for DA, the absorbance of α-Toc• at 428 nm decreased with increasing concentrations of CAs (data are not shown). The generation of α-Toc• was suppressed remarkably under the coexistence of CAs.

nol-10) show very high free-radical-scavenging activity, as listed in Table 1.22 In fact, the rate constant (ks) of 6-OHDA is 98.4 and 22.3 times larger than those of DA and 5-OHDA, respectively. As described in Section 3, the rate constants (kr) obtained for the reaction of five CAs with α-Toc• (Table 2) increased in the order given in eq 7. The kr values for DA, 5-OHDA, and 6OHDA are 2.35 × 102, 9.55 × 103, and 2.03 × 105 M−1 s−1, respectively. 6-OHDA shows very fast α-TocH-regeneration activity. The kr value of 6-OHDA is 864 and 21.3 times larger than those of DA and 5-OHDA, respectively, having the same alkyl side chain. 4.2. Comparison of Aroxyl-Scavenging and α-TocHRegeneration Rates (ks and kr) of Five CAs with Other Natural Antioxidants in 2-Propanol/Water Solution. αTocH is well known as one of the most important lipophilic AOHs in foods and biological systems.2,26,37,38 The antioxidant action of α-TocH has been ascribed to the scavenging reaction of LOO•, producing the corresponding α-Toc• radical (reaction 8). On the other hand, if α-TocH exists in biomembranes and edible oils, α-Toc• radicals may react with unsaturated lipids (LHs) (reaction 9). Reaction 9 is known as a pro-oxidant reaction, which induces degradation of LHs39−41

4. DISCUSSION 4.1. Structure−Activity Relationship in the AroxylScavenging and α-TocH-Regeneration Reactions by Five CAs and Two Catechins in 2-Propanol/Water Solution. As described in Section 3, the rate constants (ks (ArO•)) obtained for the reaction of ArO• radical with five CAs and two catechins (Table 1) increased in the order given in eq 4. Kawashima et al.13 measured the rate constants (ks (Galv•)) for the reaction of galvinoxyl (Galv•) radical with CAs (NE, EN, and DA). The ks (Galv•) values obtained are 8.1, 9.1, and 23 M−1 s−1 for NE, EN, and DA, respectively, in CH3CN at 25 °C. Furthermore, measurements of oxidation potentials (Ep) in CH3CN and DFT calculations of the highest occupied molecular orbital (HOMO) energy levels (EHOMO) of NE, EN, and DA were also performed. The results indicate that CA with lower oxidation potential (Ep) and higher HOMO energy level has higher Galv•-scavenging activity, as observed for the reaction of catechins with ArO•.21,24 Although the ks (ArO•) values obtained for ArO• (Table 1) are larger than the corresponding ks (Galv•) values for Galv•, the ratios of the rate constants (ks (ArO•)/ks (Galv•) = 2.7, 2.7, and 2.1) for NE, EN, and DA, respectively, are similar to each other. Tea catechins (EC, epicatechin gallate (ECG), epigallocatechin (EGC), and EGCG) are well known as representative polyphenolic AOHs. (1) EC and ECG and (2) EGC and EGCG have catechol and pyrogallol B-ring moieties, respectively, in a molecule. In a previous study, we reported that the rate constants (ks (ArO•)) for the reaction of (2) EGC and EGCG with ArO• radical are larger than those of (1) EC and ECG in ethanol solution, indicating that the pyrogallol Bring moiety has higher ability than that of the catechol one in scavenging ArO•.24 As shown in Figure 1, DA, NE, EN, and EC have a catechol moiety in a molecule, and 5-OHDA and EGCG have a pyrogallol moiety in a molecule. In fact, the ks values of 5-OHDA and EGCG were larger than those of DA, NE, EN, and EC (see Table 1). DA, 5-OHDA, and 6-OHDA have the same alkyl side chain (−CH2−CH2−NH2) in a molecule. The ks values obtained for DA, 5-OHDA, and 6-OHDA are 4.91 × 10, 2.17 × 102, and 4.83 × 103 M−1 s−1, respectively. DA and 5-OHDA have catechol and pyrogallol moieties in a molecule, respectively, and the rate constant (ks) of the latter is 4.42 times larger than that of the former, as observed for catechins (EC, ECG, EGC, and EGCG).24 On the other hand, 6-OHDA is considered to be a p-hydroquinone derivative, although 6-OHDA also has a catechol moiety in a molecule. Generally, biological phydroquinone derivatives (such as ubiquinol-10 and plastoqui-

k inh

LOO• + α ‐TocH ⎯→ ⎯ LOOH + α ‐Toc•

(8)

kp

α ‐Toc• + LH → α ‐TocH + L•

(9)

In a previous study, measurements of the ks and kr values were performed for representative lipid- and water-soluble AOHs (such as α-TocH, UQ10H2, Vit C (where Vit C is Na+AsH−)) in 2-PrOH/H2O solution.31 The ks and kr values reported for these AOHs are listed in Tables 1 and 2, respectively, together with those obtained in the present study. The ks and kr values of AOHs (five CAs, two catechins, αTocH, UQ10H2, Vit C, and LHs) increased in the orders given in eqs 10 and 11, respectively. LHs ≪ NE < EN < DA < EC < 5‐OHDA < EGCG < 6‐OHDA < α ‐TocH ∼ UQ 10H 2 < Vit C

(10)

LHs ≪ NE < EN < DA < (EC) < 5‐OHDA < (EGCG) < 6‐OHDA ∼ UQ 10H 2 < Vit C

(11) −1

The ks value of DA (4.91 × 10 M s ) (i.e., ArO•scavenging activity) is 2−3 orders of magnitude smaller than that of α-TocH, UQ10H2, and Vit C in 2-PrOH/H2O solution. On the other hand, the ks value of 6-OHDA (4.83 × 103 M−1 s−1), which is the largest among the five CAs studied, is comparable to those of α-TocH, UQ10H2, and Vit C. 6-OHDA showed very fast ArO•-scavenging rate (ks) (i.e., very high antioxidant activity) in 2-PrOH/H2O solution. A similar result was obtained for α-TocH-regeneration rate constants (kr) of AOHs (see Table 2). The kr value of DA (2.35 × 102 M−1 s−1) is 3−4 orders of magnitude smaller than those of UQ10H2 and Vit C, respectively. On the other hand, the kr value of 6-OHDA (2.03 × 105 M−1 s−1) is almost the same as that of UQ10H2 and 5.86 times smaller than that of Vit C. 6OHDA showed very high α-TocH-regeneration rate (kr) in 2PrOH/H2O solution. The reaction rate constants (kp) of α-Toc• with LHs (ethyl linoleate, ethyl linolenate, and ethyl arachidonate) are 8.33 × 10−2, 1.92 × 10−1, and 2.43 × 10−1 M−1 s−1, respectively, at 25.0 F

−1


Article

The Journal of Physical Chemistry B °C in toluene.41 Similarly, the reaction rate constant (kp) of αToc• with ethyl linoleate is 2.7 × 10−2 M−1 s−1 at 37 °C in ethanol.42 Generally, free-radical-scavenging rates of AOHs decrease with increasing polarity (i.e., dielectric constant (ε)) of the solvent used for the reaction.35 It has also been reported that scavenging rates remarkably decrease by hydrogen bond formation between AOHs and the solvent molecules.43 Consequently, the kp values in a polar 2-PrOH/H2O solution will be smaller than those in toluene. The kr values of DA, NE, and EN are 2.35 × 102, 7.67 × 10, and 1.68 × 102 M−1 s−1, respectively, in 2-PrOH/H2O solution (Table 2) and thus are at least 2−3 orders of magnitude larger than that (kp = 2.43 × 10−1 M−1 s−1) of ethyl arachidonate, which has the largest kp value among LHs. The result suggests that these CAs may function as AOHs by protecting the prooxidant effect of α-TocH in biological systems (such as brain and plasma). On the other hand, measurements of the rate constant (ks) for the reaction of ArO• radical with LHs have not been performed. However, we may expect that the ks values of DA, NE, and EN are also much larger than those of LHs, as observed for the kr values. Oxidative damage has been reported to play a key role in neurodegeneration and in the peripheral tissues of Parkinson’s disease patients because high levels of polyunsaturated fatty acids are present in the brain.44,45 The present results suggest that DA, NE, and EN function as AOHs in the brain tissues, by scavenging free radicals. 4.3. Suppression of the Pro-oxidant Effect of α-TocH under the Coexistence of α-TocH and Five CAs. Recently, kinetic studies of the antioxidant action of CAs (DA and LDOPA) on the peroxidation of methyl linoleate (MeLin) dispersed in Triton X-100 micelles were performed by JodkoPiόrecka and Litwinienko.16 DA and L-DOPA do not cause effective inhibition of lipid peroxidation in the MeLin/Triton X-100 micellar system. Their activity is limited to retardation of lipid peroxidation at pH 4.0−7.0 and acceleration of lipid peroxidation at pH 9.0−10.0 (i.e., they act as pro-oxidants). On the other hand, the notable suppression of lipid peroxidation was observed for the solution containing CAs and the α-TocH model (PMHC). For example, a mixture of a 1 μM α-TocH model with 10 μM L-DOPA causes 18-fold elongation of suppression time as compared to that of the 1 μM α-TocH model used alone. It has been suggested that the solution containing CAs and the α-TocH model efficiently enhances the protection of biological systems from oxidative stress. The regeneration reaction of the α-TocH model with DA may contribute to the synergistic effect of the α-TocH model and DA. As described above, α-Toc• is a key radical, which appears during the antioxidant and pro-oxidant actions of α-TocH (see reactions 2, 8, and 9). In the present study, the reaction of ArO• radical with α-TocH was performed under the coexistence of five CAs. As shown in Figure 4, the formation of α-Toc• radical was suppressed remarkably under the coexistence of α-TocH and CAs. It has been directly ascertained that the α-Toc• radical produced by the reaction with the ArO• radical immediately disappears through the regeneration reaction with DA by observing the decrease in the absorption of α-Toc• radical at 428 nm. Similar results were obtained for all the CAs studied in the present study. In fact, the α-TocH-regeneration rate constants (kr) obtained for CAs were large, as listed in Table 2. Such a direct observation of the disappearance of α-Toc• radical under the coexistence of α-TocH and five CAs has not

been reported, to our knowledge. The result indicates that the pro-oxidant effect of α-Toc• is suppressed by the coexistence of α-TocH and CAs. The concentrations of CAs (DA, NE, and EN) and α-TocH in human blood were reported by Tsakiris et al.46 The concentrations of CAs ([DA] = 55 pM, [NE] = 1.53 nM, and [EN] = 230 pM) are lower than those of α-TocH ([α-TocH] = 18.3 μM) in human blood. Consequently, the contribution of CAs to free-radical scavenging is considered to be much smaller than that of α-TocH in human blood. On the other hand, CAs are inhomogeneously distributed in the nervous tissue, and they are found in considerably high concentrations in some areas of the brain. For example, the average physiological concentration of DA ([DA]) in the human brain is ca. 8 μM.47,48 The concentration of DA in the striatum reaches 50 μM.15 DA and NE concentrations in mouse brain are 8.14 and 2.17 μM, respectively.48 Exceptionally high levels of CAs inside the synaptic vesicles (even 250 mM) and in the synaptic cleft (ca. 25 mM) are expected during neurotransmitter release.49 On the other hand, the concentration of α-TocH ([α-TocH]) in the human brain is 30 μM, and similar concentrations of α-TocH were reported for patients with Alzheimer’s disease and fetuses with Down’s syndrome.50 As described above, CAs and α-TocH coexist in nervous systems (especially in brain) at high concentrations. Consequently, we may expect a synergistic effect between CAs (DA, NE, and EN) and α-TocH in the above systems. As described in Section 1, DA, NE, and EN function as AOHs in nervous systems.6−11 The result obtained in the present kinetic study also suggests that DA, NE, and EN act as free-radical scavengers. As described above, 6-OHDA shows 2−4 orders of magnitude higher free-radical-scavenging rates (ks and kr) than those of DA, NE, and EN. 5-OHDA also shows 1−2 orders of magnitude higher scavenging rates than those of DA, NE, and EN. Consequently, we may expect that 5-OHDA and 6-OHDA also play a role as AOHs in the nervous systems. On the other hand, it has been reported that Vit C (ascorbate) is present in high concentrations in rat brain tissues; for example, the concentrations of Vit C in neurons, glia, and hippocampus are 10, 0.9, and 0.29 mM, respectively.51,52 If the CAs coexist with Vit C in tissues, the rate of regeneration of α-Toc• is represented by eq 12. The αTocH-regeneration reaction 2 of CAs may compete with that of Vit C −d[α ‐Toc•]/dt = k r CA[CA][α ‐Toc•] + k r Vit C[Vit C][α ‐Toc•]

(12)

As listed in Table 2, measurements of the second-order rate constants (krAOH) for the reaction of α-Toc• radical with five CAs and related compounds were performed in 2-PrOH/H2O solution. A large krVit C value was observed for Vit C (sodium ascorbate). The result suggests that Vit C having a large krVit C value and high concentration may contribute to the α-TocHregeneration reaction in the above brain tissues (such as neurons, glia, and hippocampus). As described above, the existence of CAs in various brain tissues has been reported, suggesting that CAs also contribute to α-TocH-regeneration reaction in some areas of the brain, although the krCA values of CAs are smaller than that of Vit C. The discussion using the concentrations of CAs and Vit C determined for the same tissue at the same time will be G



■

desirable. However, to our regret, such an example has not been reported, as far as we know. In the present study, measurements of the krCA values of CAs were performed in 2-PrOH/H2O solution. On the other hand, the krCA values of CAs will remarkably increase in biological systems (pH 7.4) due to the dissociation of OH protons in CA molecules, as observed for catechins (EC, EGC, ECG, and EGCG) having a catechol (or pyrogallol) moiety in a molecule.21,24 Measurements of krCA values of CAs in micellar solution (pH 7.4) will also be desirable. It has been reported that 5- and 6-OHDA are present in rat urine53 and also in the human caudate nucleus.54 As described in Section 1, 6-OHDA is known as a potent neurotoxin which destroys sympathetic nerves.18,19 6-OHDA likely destroys neurons by generating reactive oxygen species such as superoxide radical. It seems that 6-OHDA acts as a pro-oxidant in nervous tissues. In fact, the main use of 6-OHDA in scientific research is to induce Parkinsonism in laboratory animals, to develop new medicines for Parkinson’s disease.20 As described in a previous section, α-TocH acts as antioxidant2,26,37,38 and pro-oxidant39−41 in foods and biological systems. Similarly, it seems that 6-OHDA also has two different properties as antioxidant and pro-oxidant. However, more detailed study will be necessary to clarify the function of 6-OHDA in nervous tissues.

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 81-89-927-9588. Fax: 81-89-927-9590. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We are very grateful to Mr. Yuki Bandoh of Ehime University for the measurement of the ks values of EC and EGCG in 2PrOH/H2O solution. This work was partly supported by JSPS KAKENHI Grant Number 15k07431.

■

REFERENCES

(1) Niki, E. Assessment of antioxidant capacity in vitro and in vivo. Free Radical Biol. Med. 2010, 49, 503−515. (2) Niki, E. Role of vitamin E as a lipid-soluble peroxyl radical scavenger: in vitro and in vivo evidence. Free Radical Biol. Med. 2014, 66, 3−12. (3) Barnham, K. J.; Masters, C. L.; Bush, A. I. Neurodegenerative diseases and oxidative stress. Nat. Rev. Drug Discovery 2004, 3, 205− 214. (4) Ames, B. N.; Shigenaga, M. K.; Hagen, T. M. Oxidants, antioxidants, and the degenerative diseases of aging. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 7915−7922. (5) Iversen, S. D.; Iversen, L. L. Dopamine: 50 years in perspective. Trends Neurosci. 2007, 30, 188−193. (6) Troadec, J.-D.; Marien, M.; Darios, F.; Hartmann, A.; Ruberg, M.; Colpaert, F.; Michel, P. P. Noradrenaline provides long-term protection to dopaminergic neurons by reducing oxidative stress. J. Neurochem. 2001, 79, 200−210. (7) Iacovitti, L.; Stull, N. D.; Mishizen, A. Neurotransmitters, KCl and antioxidants rescue striatal neurons from apoptotic cell death in culture. Brain Res. 1999, 816, 276−285. (8) Noh, J. S.; Kim, E. Y.; Kang, J. S.; Kim, H. R.; Oh, Y. J.; Gwag, B. J. Neurotoxic and neuroprotective actions of catecholamines in cortical neurons. Exp. Neurol. 1999, 159, 217−224. (9) Colamartino, M.; Padua, L.; Meneghini, C.; Leone, S.; Cornetta, T.; Testa, A.; Cozzi, R. Protective effects of L-dopa and carbidopa combined treatments on human catecholaminergic cells. DNA Cell Biol. 2012, 31, 1572−1579. (10) Cornetta, T.; Palma, S.; Aprile, L.; Padua, L.; Tonali, P.; Testa, A.; Cozzi, R. Levodopa therapy reduces DNA damage in peripheral blood cells of patients with Parkinson’s disease. Cell Biol. Toxicol. 2009, 25, 321−330. (11) Cosentino, M.; Rasini, E.; Colombo, C.; Marino, F.; Blandini, F.; Ferrari, M.; Samuele, A.; Lecchini, S.; Nappi, G.; Frigo, G. Dopaminergic modulation of oxidative stress and apoptosis in human peripheral blood lymphocytes: evidence for a D1-like receptor-dependent protective effect. Free Radical Biol. Med. 2004, 36, 1233−1240. (12) Cosa, G.; Scaiano, J. C. Reactivity of adrenaline toward alkoxyl radicals and carbonyl triplet states. Org. Biomol. Chem. 2008, 6, 4609− 4614. (13) Kawashima, T.; Ohkubo, K.; Fukuzumi, S. Radical scavenging reactivity of catecholamine neurotransmitters and the inhibition effect for DNA cleavage. J. Phys. Chem. B 2010, 114, 675−680. (14) Ohkubo, K.; Moro-oka, Y.; Fukuzumi, S. Hydrogen abstraction from neurotransmitters by active oxygen species facilitated by intramolecular hydrogen bonding in the radical intermediates. Org. Biomol. Chem. 2006, 4, 999−1001. (15) Liu, J.; Mori, A. Monoamine metabolism provides an antioxidant defense in the brain against oxidant- and free radicalinduced damage. Arch. Biochem. Biophys. 1993, 302, 118−127. (16) Jodko-Piόrecka, K.; Litwinienko, G. Antioxidant activity of dopamine and L-DOPA in lipid micelles and their cooperation with an analogue of α-tocopherol. Free Radical Biol. Med. 2015, 83, 1−11.

5. CONCLUSIONS DA, NE, and EN are well known as CA neurotransmitters. 5and 6-OHDA are the oxidation products of DA. These compounds contain two or three phenolic OH groups in a molecule and may function as AOHs in nervous systems (especially in brain). However, kinetic studies on the freeradical-scavenging activity of CAs are very limited.12−14 In the present study, measurements of ArO•-scavenging and α-TocHregeneration rate constants (ks and kr) of five CAs and two catechins (EC and EGCG) were performed in 2-PrOH/H2O solution at 25.0 °C, using single- and double-mixing stoppedflow spectrophotometry, respectively. Both the ks and kr values increased in the order of LHs ≪ NE < EN < DA < EC < 5OHDA < EGCG < 6-OHDA. The ks and kr values of NE, EN, and DA are smaller than (or comparable to) that of EC. However, the ks and kr values of NE, EN, and DA are at least 2−3 orders of magnitude larger than those of LHs. 6-OHDA showed fast ArO•-scavenging and α-Toc•-regeneration rates similar (or comparable) to those of UQ10H2 and Na+AsH−, which are well known as representative lipid- and water-soluble AOHs. The results suggest that the CAs may contribute to protection from oxidative damage in nervous tissues by scavenging free radicals (such as LOO• and LO•) and regenerating α-TocH from the α-Toc• radical. The UV−vis absorption of α-Toc•, which has been produced by the reaction of α-TocH with ArO•, efficiently disappeared under the existence of CAs due to the above fast regeneration reaction. The result indicates that the pro-oxidant effect of αToc• is suppressed by the coexistence of CAs. As α-TocH and CAs coexist in relatively high concentrations in nervous systems, the above synergistic effect, that is, the suppression of the pro-oxidant reaction of α-Toc• by CAs, may function in the systems. H


Article


edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one). Bull. Chem. Soc. Jpn. 2006, 79, 421−426. (37) 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. (38) Neužil, J.; Thomas, S. R.; Stocker, R. Requirement for, promotion, or inhibition by α-tocopherol of radical induced initiation of plasma lipoprotein lipid peroxidation. Free Radical Biol. Med. 1997, 22, 57−71. (39) Terao, J.; Matsushita, S. The peroxidizing effect of α-tocopherol on autoxidation of methyl linoleate in bulk phase. Lipids 1986, 21, 255−260. (40) 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. (41) 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. (42) Watanabe, A.; Noguchi, N.; Fujisawa, A.; Kodama, T.; Tamura, K.; Cynshi, O.; Niki, E. Stability and reactivity of aroxyl radicals derived from a novel antioxidant BO-653 and related compounds. Effects of substituent and side chain in solution and membranes. J. Am. Chem. Soc. 2000, 122, 5438−5442. (43) Avila, D. V.; Ingold, K. U.; Lusztyk, J.; Green, W. H.; Procopio, D. R. Dramatic solvent effects on the absolute rate constants for abstraction of the hydroxylic hydrogen atom from tert-butyl hydroperoxide and phenol by the cumyloxyl radical. The role of hydrogen bonding. J. Am. Chem. Soc. 1995, 117, 2929−2930. (44) Mariani, E.; Polidori, M. C.; Cherubini, A.; Mecocci, P. Oxidative stress in brain aging, neurodegenerative and vascular diseases: An overview. J. Chromatogr. B 2005, 827, 65−75. (45) Sanders, L. H.; Greenamyre, J. T. Oxidative damage to macromolecules in human Parkinson disease and the rotenone model. Free Radical Biol. Med. 2013, 62, 111−120. (46) Tsakiris, S.; Karikas, G. A.; Parthimos, T.; Tsakiris, T.; Bakogiannis, C.; Schulpis, K. H. Alpha-tocopherol supplementation prevents the exercise-induced reduction of serum paraoxonase 1/ arylesterase activities in healthy individuals. Eur. J. Clin. Nutr. 2009, 63, 215−221. (47) Musshoff, F.; Lachenmeier, D. W.; Schmidt, P.; Dettmeyer, R.; Madea, B. Systematic regional study of dopamine, norsalsolinol, and (R/S)-salsolinol levels in human brain areas of alcoholics. Alcohol.: Clin. Exp. Res. 2005, 29, 46−52. (48) Naka, F.; Shiga, T.; Yaguchi, M.; Okado, N. An enriched environment increases noradrenaline concentration in the mouse brain. Brain Res. 2002, 924, 124−126. (49) Milutinovic, P. S.; Yang, L.; Cantor, R. S.; Eger, E. I.; Sonner, J. M. Anesthetic-like modulation of a γ-aminobutyric acid type A, strychnine-sensitive glycine, and N-methyl-D-aspartate receptors by coreleased neurotransmitters. Anesth. Analg. 2007, 105, 386−392. (50) Metcalfe, T.; Bowen, D. M.; Muller, D. P. R. Vitamin E concentrations in human brain of patients with Alzheimer’s disease, fetuses with down’s syndrome, centenarians, and controls. Neurochem. Res. 1989, 14, 1209−1212. (51) Rice, M. E.; Russo-Menna, I. Differential compartmentalization of brain ascorbate and glutathione between neurons and glia. Neuroscience 1997, 82, 1213−1223. (52) Ferreira, N. R.; Santos, R. M.; Laranjinha, J.; Barbosa, R. M. Real time in vivo measurement of ascorbate in the brain using carbon nanotube-modified microelectrodes. Electroanalysis 2013, 25, 1757− 1763. (53) Andrew, R.; Watson, D. G.; Best, S. A.; Midgley, J. M.; Wenlong, H.; Petty, R. K. H. The determination of hydroxydopamines and other trace amines in the urine of Parkinsonian patients and normal controls. Neurochem. Res. 1993, 18, 1175−1177. (54) Curtius, H. Ch.; Wolfensberger, M.; Steinmann, B.; Redweik, U.; Siegfried, J. Mass fragmentography of dopamine and 6-

(17) Lin, C.-M.; Lin, Y.-T.; Lin, R.-D.; Huang, W.-J.; Lee, M.-H. Neurocytoprotective effects of aliphatic hydroxamates from lovastatin, a secondary metabolite from Monascus-fermented red mold rice, in 6hydroxydopamine (6-OHDA)-treated nerve growth factor (NGF)differentiated PC12 cells. ACS Chem. Neurosci. 2015, 6, 716−724. (18) Kostrzewa, R. M.; Jacobwitz, D. M. Pharmacological actions of 6-hydroxydopamine. Pharmacol. Rev. 1974, 26, 199−288. (19) Hanrott, K.; Gudmunsen, L.; O’Neill, M. J.; Wonnacott, S. 6Hydroxydopamine-induced apoptosis is mediated via extracellular auto-oxidation and caspase 3-dependent activation of protein kinase Cδ. J. Biol. Chem. 2005, 281, 5373−5382. (20) Breese, G. R.; Knapp, D. J.; Criswell, H. E.; Moy, S. S.; Papadeas, S. T.; Blake, B. L. The neonate-6-hydroxydopamine-lesioned rat: a model for clinical neuroscience and neurobiological principles. Brain Res. Rev. 2005, 48, 57−73. (21) Mukai, K.; Mitani, S.; Ohara, K.; Nagaoka, S. Structure-activity relationship of the tocopherol-regeneration reaction by catechins. Free Radical Biol. Med. 2005, 38, 1243−1256. (22) Mukai, K.; Itoh, S.; Morimoto, H. Stopped-flow kinetic study of vitamin E regeneration reaction with biological hydroquinones (reduced forms of ubiquinone, vitamin K, and tocopherolquinone) in solution. J. Biol. Chem. 1992, 267, 22277−22281. (23) Mukai, K.; Tokunaga, A.; Itoh, S.; Kanesaki, Y.; Ohara, K.; Nagaoka, S.; Abe, K. Structure-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. (24) 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. (25) 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. (26) Traber, M. G.; Atkinson, J. Vitamin E, antioxidant and nothing more. Free Radical Biol. Med. 2007, 43, 4−15. (27) 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. (28) Lass, A.; Forster, M. J.; Sohal, R. S. Effects of coenzyme Q10 and α-tocopherol administration on their tissue levels in the mouse: Elevation of mitochondrial α-tocopherol by coenzyme Q10. Free Radical Biol. Med. 1999, 26, 1375−1382. (29) 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. (30) Colomé, 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. (31) 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. (32) Rieker, A.; Scheffler, K. Die beteiligung von phenylresten an der aroxylmesomerie. Liebigs Ann. Chem. 1965, 689, 78−92. (33) Ouchi, A.; Nagaoka, S.; Mukai, K. Tunneling effect in regeneration reaction of vitamin E by ubiquinol. J. Phys. Chem. B 2010, 114, 6601−6607. (34) Jovanovic, S. V.; Hara, Y.; Steenken, S.; Simic, M. G. Antioxidant potential of gallocatechins. A pulse radiolysis and laser photolysis study. J. Am. Chem. Soc. 1995, 117, 9881−9888. (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) Ohara, K.; Fujii, A.; Ichimura, Y.; Sato, K.; Mukai, K. Kinetic study of radical-scavenging and vitamin E-regenerating actions of I


Article

The Journal of Physical Chemistry B hydroxydopamine: Application to the determination of dopamine in human brain biopsies from the caudate nucleus. J. Chromatogr. 1974, 99, 529−540.

J


Kinetic Study of Aroxyl-Radical-Scavenging and Î±-Tocopherol

Recommend Documents