Effects of reaction environments on radical-scavenging mechanisms of ascorbic acid

Abstract

The effects of reaction environments on the radical-scavenging mechanisms of ascorbic acid (AscH₂) were investigated using 2,2-diphenyl-1-picrylhydrazyl radical (DPPH^•) as a reactivity model of reactive oxygen species. Water-insoluble DPPH^• was solubilized by β-cyclodextrin (β-CD) in water. The DPPH^•-scavenging rate of AscH₂ in methanol (MeOH) was much slower than that in phosphate buffer (0.05 M, pH 7.0). An organic soluble 5,6-isopropylidene-l-ascorbic acid (iAscH₂) scavenged DPPH^• much slower in acetonitrile (MeCN) than in MeOH. In MeOH, Mg(ClO₄)₂ significantly decelerated the DPPH^•-scavenging reaction by AscH₂ and iAscH₂, while no effect of Mg(ClO₄)₂ was observed in MeCN. On the other hand, Mg(ClO₄)₂ significantly accelerated the reaction between AscH₂ and β-CD-solubilized DPPH^• (DPPH^•/β-CD) in phosphate buffer (0.05 M, pH 6.5), although the addition of 0.05 M Mg(ClO₄)₂ to the AscH₂–DPPH^•/β-CD system in phosphate buffer (0.05 M, pH 7.0) resulted in the change in pH of the phosphate buffer to be 6.5. Thus, the DPPH^•-scavenging reaction by iAscH₂ in MeCN may proceed via a one-step hydrogen-atom transfer, while an electron-transfer pathway is involved in the reaction between AscH₂ and DPPH^•/β-CD in phosphate buffer solution. These results demonstrate that the DPPH^•-scavenging mechanism of AscH₂ are affected by the reaction environments.

Introduction

Hydrogen-transfer reactions are of considerable importance as an initial step of the radical-scavenging reaction of antioxidants. It is known that there are several reaction mechanisms for the hydrogen-transfer reactions from antioxidants (AH) to radicals (R^•), such as one-step hydrogen-atom transfer (HAT), electron transfer followed by proton transfer (ET–PT), and sequential proton-loss electron transfer (SPLET) as shown in Fig. 1.⁽¹⁾ It is also known that the radical-scavenging reactivity and mechanism are significantly affected by the reaction environments, such as solvents,^(2,3) pH,^(4,5) the presence of metal ions,^(6–14) and so on. In fact, we reported that the radical-scavenging reaction of (+)-catechin is significantly accelerated in the presence of magnesium ion (Mg²⁺) in acetonitrile (MeCN), an aprotic polar solvent.⁽⁶⁾ Because electron-transfer reactions are known to be accelerated in the presence of redox inactive metal ions, such as Mg²⁺,^(15,16) the radical-scavenging reaction of (+)-catechin proceeds via the ET–PT mechanism (Fig. 1), where the coordination of Mg²⁺ to the one-electron reduced species of the radical, the corresponding anion (R⁻), may stabilize the product, resulting in the acceleration of electron transfer.⁽⁶⁾ Furthermore, we also reported that a vitamin E model compound scavenges radicals via the HAT mechanism in MeCN, while this reaction proceeds via the ET–PT mechanism in methanol (MeOH), a protic polar solvent.⁽⁷⁾ 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH^•) (Fig. 2) is a relatively stable radical at room temperature and has been frequently used as a reactivity model of reactive oxygen species (ROS) to evaluate the activity of antioxidants for more than 60 years.^(17–19) However, the insolubility of DPPH^• in water has precluded its use in aqueous media, especially in concentrated buffer solutions, without cosolvent, such as ethanol. Recently, we have succeeded in solubilizing DPPH^• in water using β-cyclodextrin (β-CD) [Eq. 1].^(20,21) This enables us to use DPPH^• in aqueous solutions, especially in physiological concentrations of buffer solutions, to evaluate the radical-scavenging activity as well as mechanism of water-soluble antioxidants.^(20–22) We report herein the effects of reaction environments on the radical-scavenging mechanisms of ascorbic acid (AscH₂), a representative water-soluble antioxidant, and its derivative, 5,6-isopropylidene-l-ascorbic acid (iAscH₂) (Fig. 2). iAscH₂ was used instead of AscH₂ in MeCN because of the low solubility of AscH₂ in MeCN.⁽²³⁾ The use of the same ROS model radical, DPPH^•, both in aqueous and non-aqueous media provides fundamental and valuable information about the effects of reaction environments on the radical-scavenging mechanisms of AscH₂ for the first time.

Fig. 1.

Mechanisms of radical (R^•)-scavenging reaction by antioxidants (AH). HAT, hydrogen-atom transfer; ET–PT, electron transfer followed by proton transfer; SPLET, sequential proton-loss electron transfer.

Fig. 2.

Chemical structures of AscH₂, iAscH₂, and DPPH^•.

[1]

Materials and Methods

Materials

Ascorbic acid [l(+)-ascorbic acid, AscH₂], DPPH^•, magnesium perchlorate [Mg(ClO₄)₂], and phosphate buffer solutions (0.1 M, pH 6.0, 6.5, 6.8, 7.0, 7.6, and 8.0) were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). iAscH₂ was commercially obtained from Aldrich. D₂O, MeOH (spectral grade), and MeCN (spectral grade) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan) and used as received. Sodium ascorbate (sodium l-ascorbate, NaAscH) and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO^•) was commercially obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). The water used in this study was freshly prepared with a Milli-Q system (Millipore Direct-Q UV3). The deuterated phosphate buffer solutions were prepared by dissolving phosphate buffer powder (FUJIFILM Wako Pure Chemical Corporation) to D₂O and the pD was adjusted by adding 5 N hydrochloric acid (FUJIFILM Wako Pure Chemical Corporation). The pD values were calculated by adding 0.4 to the corresponding pH values measured by a HORIBA D-51 pH meter.⁽²⁴⁾ DPPH^• was solubilized in water by β-CD (Tokyo Chemical Industry Co., Ltd.) according to the procedure reported in the literatures.^(20,21) Tetra-n-butylammonium perchlorate (Bu₄NClO₄), used as a supporting electrolyte for electrochemical measurements, was purchased from Tokyo Chemical Industry Co., Ltd., recrystallized from ethanol, and dried under vacuum at 313 K.

Spectral and kinetic measurements

The rates of DPPH^•-scavenging reactions by AscH₂ in MeOH were determined by monitoring the absorbance change at 516 nm due to DPPH^• (ɛ = 1.13 × 10⁴ M⁻¹ cm⁻¹) after mixing of DPPH^• in MeOH with an MeOH solution containing AscH₂ at a volumetric ratio of 1:1 using a stopped-flow technique on a UNISOKU RSP-1000-02NM spectrophotometer at 298 K. The rates in MeCN and phosphate buffer solutions were also determined in the same manner by monitoring the absorbance change at 519 nm (ɛ = 1.18 × 10⁴ M⁻¹ cm⁻¹) and 527 nm (ɛ = 1.07 × 10⁴ M⁻¹ cm⁻¹),^(20,21) respectively, due to DPPH^•. The pseudo-first-order rate constants (k_obs) were determined by a least-square curve fit using an Apple MacBook Pro personal computer. The first-order plots of ln(A – A_∞) vs time (A and A_∞ are denoted as the absorbance at the reaction time and the final absorbance, respectively) were linear until three or more half-lives with the correlation coefficient ρ>0.999.

Electrochemical measurements

The cyclic voltammetry (CV) measurements were performed on an ALS-630A electrochemical analyzer in MeOH or MeCN containing 0.10 M Bu₄NClO₄ as a supporting electrolyte or in phosphate buffer solutions (0.1 M, pH 7.0). The glassy carbon working electrode (BAS) was polished with BAS polishing alumina suspension and a polishing cloth and rinsed with Milli-Q water followed by MeOH prior to each measurement. The counter electrode was a platinum wire. The solution concentrations were 1.4 × 10⁻³ M for AscH₂, iAscH₂, and NaAscH unless otherwise noted. The measured potentials were recorded with respect to an Ag/AgCl reference electrode with the sweep rate of 100 mV s⁻¹.

Results

Upon mixing of AscH₂ with DPPH^• in MeOH on a stopped-flow spectrophotometer, the absorption band at 516 nm due to DPPH^• disappeared rapidly as shown in Fig. 3. The decay of the absorbance at 516 nm obeyed pseudo-first-order kinetics, when the concentration of AscH₂ ([AscH₂]) was maintained at more than a 10-fold excess of DPPH^• concentration (inset of Fig. 3). The pseudo-first-order rate constants (k_obs) increased with increasing [AscH₂] as shown in Fig. 4 (closed circles). When AscH₂ was replaced by iAscH₂ in MeOH, a similar result was obtained as the case of AscH₂ (Fig. 5, closed circles). When MeCN was used as the solvent instead of MeOH, the k_obs values for the DPPH^•-scavenging reaction by iAscH₂ were much slower than those in MeOH (Fig. 6). In contrast to the case in MeOH, the plot of k_obs vs [iAscH₂] gave a straight line passing through the origin (closed squares in Fig. 6). From the slope of the linear plot, the second-order rate constant (k_H) for the DPPH^•-scavenging reaction by iAscH₂ was determined in MeCN to be 1.3 × 10 M⁻¹ s⁻¹.

Fig. 3.

Spectral change (interval: 0.2 s) observed during the reaction of AscH₂ (1.0 × 10⁻² M) with DPPH^• (6.8 × 10⁻⁵ M) in MeOH at 298 K. Inset: the first-order plot of the absorbance at 516 nm.

Fig. 4.

Plots of k_obs vs [AscH₂] in the absence (closed circles) and presence (open circles) of Mg(ClO₄)₂ in MeOH.

Fig. 5.

Plots of k_obs vs [iAscH₂] in the absence (closed circles) and presence (open circles) of Mg(ClO₄)₂ in MeOH.

Fig. 6.

Plots of k_obs vs [iAscH₂] in the absence (closed squares) and presence (open circles) of Mg(ClO₄)₂ in MeCN.

If the electron-transfer reaction is involved in the radical-scavenging reaction as the rate-determining step, the radical-scavenging rate would be accelerated in the presence of redox inactive metal ions.^(6–10) Thus, the effect of a redox inactive metal ion on the reaction between AscH₂ and DPPH^• was investigated with use of Mg(ClO₄)₂. When 0.05 M Mg(ClO₄)₂ was added to the AscH₂–DPPH^• system in MeOH, the k_obs values were significantly decreased as shown in Fig. 4 (open circles). A similar result was obtained for the iAscH₂–DPPH^• system in the presence of 0.05 M Mg(ClO₄)₂ in MeOH (Fig. 5, open circles). In MeCN, however, no effect of 0.05 M Mg(ClO₄)₂ on the k_obs value for the reaction between iAscH₂ and DPPH^• was observed as shown in Fig. 6 (open circles).

The reaction between AscH₂ and β-CD-solubilized DPPH^• (DPPH^•/β-CD) was also investigated in a phosphate buffer (0.05 M, pH 7.0). Upon mixing of DPPH^•/β-CD in water (Milli-Q) with a phosphate buffer solution (0.1 M, pH 7.0) of AscH₂ at a volumetric ratio of 1:1 on a stopped-flow spectrophotometer, the absorption band at 527 nm due to DPPH^• rapidly decreased as shown in Fig. 7. Since the pK_a value of AscH₂ is 4.1 and thus AscH₂ undergoes deprotonation to produce ascorbate anion (AscH⁻) in phosphate buffer solution (0.05 M, pH 7.0),^(25,26) this spectral change indicates that AscH⁻ efficiently scavenged DPPH^• [Eq. 2]. The decay of the absorbance at 527 nm monitored by a stopped-flow technique obeyed pseudo-first-order kinetics, when [AscH₂] was maintained at more than a 10-fold excess of the concentration of DPPH^•/β-CD. The k_obs values in phosphate buffer solution were much larger than those in MeOH. The k_obs values linearly increased with increasing [AscH₂] passing through the origin (Fig. 8, closed circles). From the slope of the linear plot, the k_H value for the scavenging reaction of DPPH^•/β-CD by AscH₂ was determined to be 5.6 × 10³ M⁻¹ s⁻¹ in phosphate buffer solution (0.05 M, pH 7.0) at 298 K.

Fig. 7.

Spectral change (interval: 30 ms) observed during the reaction of AscH₂ (1.4 × 10⁻³ M) with DPPH^•/β-CD (3.4 × 10⁻⁵ M) in phosphate buffer solution (0.05 M, pH 7.0) at 298 K. Inset: the first-order plot of the absorbance at 527 nm.

Fig. 8.

Plots of k_obs vs [AscH₂] in phosphate buffer (0.05 M, pH 7.0) (closed circles), phosphate buffer (0.05 M, pH 6.5) (closed squares), phosphate buffer (0.05 M, pH 6.5) containing 0.05 M Mg(ClO₄)₂ (open circles), and deuterated phosphate buffer (0.05 M, pD 7.0) (closed triangles).

[2]

Figure 9 shows the plot of the k_H values vs pH of phosphate buffer solutions (0.05 M). The k_H values increase with pH (Table 1).

Fig. 9.

Plot of k_H vs pH.

Table 1.

Second-order rate constants (k_H and k_D) for the reaction of AscH₂ with DPPH^•/β-CD in 0.05 M phosphate buffer solutions at 298 K

pH or pD	kH or kD (M−1 s−1)
6.0	3.7 × 103
6.5	3.9 × 103
6.5 + 0.05 M Mg(ClO4)2	9.5 × 103
6.5 (pD)	1.2 × 103 (kD)
6.8	5.4 × 103
7.0	5.6 × 103
7.0 (pD)	1.5 × 103 (kD)
7.2	7.4 × 103
7.6	1.3 × 104
8.0	2.2 × 104

When 0.05 M Mg(ClO₄)₂ was added to the AscH₂–DPPH^•/β-CD system in phosphate buffer solution (0.05 M, pH 7.0), the k_H value significantly increased to be 9.5 × 10³ M⁻¹ s⁻¹ as shown in Fig. 8 (open circles). On the other hand, the pH of the phosphate buffer solution (pH 7.0) decreased to be 6.5 upon addition of 0.05 M Mg(ClO₄)₂. The k_H value at pH 6.5 without Mg(ClO₄)₂ is 3.9 × 10³ M⁻¹ s⁻¹ (closed squares in Fig. 8). Thus, the DPPH^•-scavenging reaction by AscH₂ in the presence of 0.05 M Mg(ClO₄)₂ was more than 2-fold faster than that in its absence. The k_H value at pH 7.0 in the presence of 0.05 M Mg(ClO₄)₂ could not be determined due to the poor solubility of Mg(ClO₄)₂ in phosphate buffer at pH 7.0.

D₂O was used to prepare a phosphate buffer instead of H₂O to investigate the kinetic isotope effect (KIE) in the scavenging reaction of DPPH^•/β-CD by AscH₂. In the presence of D₂O, the exchangeable O–H protons are replaced by deuterons from D₂O. In a deuterated buffer solution (0.05 M, pD 7.0), the second-order rate constant (k_D) for the reaction between AscD₂ and DPPH^•/β-CD was determined to be 1.5 × 10³ M⁻¹ s⁻¹, which is significantly smaller than the corresponding k_H value (5.6 × 10³ M⁻¹ s⁻¹) as shown in Fig. 8 and Table 1. Thus, the KIE (k_H/k_D) is calculated to be 3.7. The KIE of 3.3 was also observed at pH 6.5.

The solvent effect of on the electron-donor ability of AscH₂ and iAscH₂ was examined by CV measurements. An irreversible oxidation (anodic) peak was observed for AscH₂ in MeOH containing 0.10 M Bu₄NClO₄ used as a supporting electrolyte as shown in Fig. 10. From the cyclic voltammogram, the oxidation peak potential (E_pa) of AscH₂ in MeOH (0.10 M Bu₄NClO₄) was determined to be +0.89 V vs Ag/AgCl at the scan rate of 100 mV s⁻¹. The E_pa values of AscH₂ and iAscH₂, which were determined from the cyclic voltammograms (Fig. 10) at the scan rate of 100 mV s⁻¹ in various solvents are listed in Table 2. The E_pa value of iAscH₂ in MeOH (0.10 M Bu₄NClO₄) (+0.87 V vs Ag/AgCl) was about the same as that of AscH₂. In MeCN (0.10 M Bu₄NClO₄), the E_pa value of iAscH₂ was largely shifted to the positive direction (+1.24 V vs Ag/AgCl) as compared to that in MeOH (0.10 M Bu₄NClO₄). On the other hand, the E_pa value of AscH₂ in phosphate buffer (0.1 M, pH 7.0) (+0.24 V vs Ag/AgCl) was significantly shifted to the negative direction as compared to that in MeOH (0.10 M Bu₄NClO₄). To determine the E_pa values of AscH⁻, NaAscH was used as a source of AscH⁻. The E_pa value of NaAscH in MeOH (0.10 M Bu₄NClO₄) (+0.13 V vs Ag/AgCl) thus obtained was largely shifted to the negative direction as compared to that of AscH₂, although the peak current was much smaller than AscH₂ (Fig. 10) because of the poor solubility of NaAscH in MeOH.⁽²⁷⁾ The E_pa value of NaAscH in phosphate buffer (0.1 M, pH 7.0) was located at +0.19 V vs Ag/AgCl, which is slightly positive than that in MeOH (0.10 M Bu₄NClO₄) (+0.13 V) and slightly negative than that of AscH₂ in phosphate buffer (0.1 M, pH 7.0) (+0.24 V).

Fig. 10.

Cyclic voltammograms of AscH₂ (1.4 × 10⁻³ M) (black lines), iAscH₂ (1.4 × 10⁻³ M) (dashed lines), and NaAscH (1.4 × 10⁻³ M in phosphate buffer) (gray lines) in MeOH (0.10 M Bu₄NClO₄), MeCN (0.10 M Bu₄NClO₄), and phosphate buffer (PB, 0.1 M, pH 7.0) recorded at the scan rate of 100 mV s⁻¹ on a glassy carbon working electrode. The concentration of NaAscH in MeOH (0.10 M Bu₄NClO₄) could not be determined precisely because of the poor solubility of NaAscH in MeOH.

Table 2.

Oxidation peak potentials (E_pa) (vs Ag/AgCl) of AscH₂, iAscH₂, and NaAscH in various solvents

Solvent	Epa(AscH2)a) (V)	Epa(iAscH2)a) (V)	Epa(NaAscH)a) (V)
Phosphate buffer (0.1 M, pH 7.0)	+0.24	—	+0.19
MeOHb)	+0.89	+0.87	+0.13
MeCNb)	—	+1.24	—

^a)Recorded with a glassy carbon working electrode at sweep rate of 100 mV s⁻¹. ^b)Containing 0.10 M Bu₄NClO₄ as a supporting electrolyte.

Discussion

As mentioned above, in phosphate buffer solutions used in this study (0.05 M, pH 6.0–8.0), AscH₂, whose pK_a value is 4.1,^(25,26) undergoes deprotonation and AscH⁻ thus produced is an active species to scavenge DPPH^•/β-CD [Eq. 2]. In fact, the E_pa value of AscH₂ in phosphate buffer (0.1 M, pH 7.0) (+0.24 V vs Ag/AgCl) is about the same as that of NaAscH (+0.19 V vs Ag/AgCl) as shown in Fig. 10 and Table 2, indicating that AscH₂ exists as AscH⁻ in the phosphate buffer solution at pH 7.0. In such a case, the plot of k_obs vs [AscH₂] showed the linear correlation passing through the origin as shown in Fig. 8. On the other hand, such a dependence was not observed for the reactions of AscH₂ and iAscH₂ with DPPH^• in MeOH (Fig. 4 and 5, respectively). In MeOH, AscH₂ may be in equilibrium with AscH⁻ as shown in Fig. 11A. Because AscH⁻ may form a complex with DPPH^• prior to the hydrogen-transfer reaction, the plot of k_obs vs [AscH₂] or [iAscH₂] would be fitted by a Michaelis–Menten type saturation dependence with use of [Eq. 3], where K is the proton dissociation constant (K_d) from AscH₂ times the formation constant (K_c) of a complex between AscH⁻ and DPPH^• (K = K_dK_c), and k_∞ is the hydrogen-transfer rate constant from AscH– to DPPH^• in the complex, as shown by the dashed lines in Fig. 4 and 5.

Fig. 11.

Proposed reaction mechanisms for the DPPH^•-scavenging reaction by AscH₂ or iAscH₂ in (A) MeOH and (B) MeCN.

k_obs = k_∞K[AscH₂ or iAscH₂]/(1 + K[AscH₂ or iAscH₂]) [3]

Hydrogen transfer from AscH⁻ to DPPH^• occurs to produce Asc^•− and DPPH-H. The k_∞ values for AscH₂ and iAscH₂ are 1.9 s⁻¹ and 2.3 s⁻¹, respectively. The K values are determined to be 2.1 × 10² M⁻¹ and 1.7 × 10² M⁻¹ for AscH₂ and iAscH₂, respectively. As mentioned above, in the phosphate buffer solutions, the addition of 0.05 M Mg(ClO₄)₂ resulted in the decrease of the pH from 7.0 to 6.5. Since the E_pa value of AscH₂ (+0.89 V vs Ag/AgCl) is much more positive than that of NaAscH used as the AscH⁻ source in MeOH (0.10 M Bu₄NClO₄) (+0.13 V vs Ag/AgCl), the electron donor ability of AscH₂ in MeOH is much lower than that in phosphate buffer (0.05 M, pH 7.0), leading to the much lower k_obs values in MeOH as compared to those in phosphate buffer (0.05 M, pH 7.0). In MeOH, the acidity of the solution may be increased in the presence of 0.05 M Mg(ClO₄)₂. In such a case, the deprotonation equilibrium in Fig. 11A may lie far to the left. That is why the rates of DPPH^•-scavenging by AscH₂ or iAscH₂ were significantly decreased in the presence of 0.05 M Mg(ClO₄)₂ in MeOH. Furthermore, a complex formation was reported between AscH⁻ and Mg²⁺.^(28,29) In fact, a shift of the absorption band at 245 nm due to AscH₂ or iAscH₂ to 249 nm was observed upon addition of 0.05 M Mg(ClO₄)₂ in MeOH (data not shown), while such a shift was not observed for AscH₂ by adding 0.05 M Mg(ClO₄)₂ in phosphate buffer solution. Such a complex formation between AscH⁻ and Mg²⁺ may preclude the complex formation between AscH⁻ and DPPH^• (Fig. 11A). The detailed investigation of the complexation of AscH⁻ with Mg²⁺ in MeOH and its effect on the rate of the DPPH^•-scavenging reaction is underway. On the other hand, the pK_a value of iAscH₂ in MeCN was reported to be 18.3.⁽³⁰⁾ Thus, little deprotonation of iAscH₂ occurs to produce iAscH⁻ in MeCN. In such a case, the DPPH^•-scavenging reaction by iAscH₂ may proceed via the HAT mechanism as the rate-determining step (Fig. 11B). In fact, no effect of Mg(ClO₄)₂ was observed on the rate of DPPH^•-scavenging in MeCN (Fig. 6). Since the E_pa value of iAscH₂ (+1.24 V vs Ag/AgCl) in MeCN (0.10 M Bu₄NClO₄) is much more positive than that of in MeOH (0.10 M Bu₄NClO₄) (+0.87 V vs Ag/AgCl), the electron donor ability of iAscH₂ in MeCN is much lower than that in MeOH. Thus, the HAT mechanism may be more feasible in MeCN.

In the phosphate buffer solutions, the significant acceleration was observed in the presence of 0.05 M Mg(ClO₄)₂ (Fig. 8). This suggests that an electron-transfer process may be involved as the rate-determining step, where the coordination of Mg²⁺ to the one-electron reduced species of DPPH^• (DPPH⁻) may stabilize the product, resulting in the acceleration of electron transfer. Furthermore, the KIEs of 3.7 and 3.3 were observed at pH/pD 7.0 and 6.5, respectively. On the other hand, we have recently reported that a large KIE (12.8) was observed for the hydrogen-transfer reaction from AscH₂ to 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO^•) in a phosphate buffer (0.05 M, pH/pD 7.0).⁽³¹⁾ Such a large KIE value has clearly precluded an electron-transfer pathway in the oxidation of AscH₂ by PTIO^•. In fact, no acceleration was observed upon addition of 0.05 M Mg(ClO₄)₂ to the AscH₂ by PTIO^• in phosphate buffer (0.05 M, pH 6.5) as shown in Fig. 12. The k_H values for the reaction between AscH₂ and PTIO^• in the absence and presence of 0.05 M Mg(ClO₄)₂ were determined from the slopes of the linear plots in Fig. 12 to be 2.4 × 10³ M⁻¹ s⁻¹ and 2.1 × 10³ M⁻¹ s⁻¹, respectively in phosphate buffer solution (0.05 M, pH 6.5). These results suggest that the hydrogen-transfer reaction from AscH₂ to DPPH^•/β-CD in phosphate buffer solution may proceed via a proton-coupled electron transfer, where the electron-transfer process is partly involved as the rate-determining step. Because the second pK_a value of AscH₂ is 11.4,^(25,26) the pH dependence of the k_H values shown in Fig. 9 is not caused by the second deprotonation of AscH₂ to produce the dianion of AscH₂ (Asc²⁻). The reason for such a pH dependence is now under investigation with use of other water-soluble antioxidants, such as Trolox, (+)-catechin, and caffeic acid, instead of AscH₂.

Fig. 12.

Plots of k_obs vs [AscH₂] for the reaction of AscH₂ with PTIO^• in the absence (closed circles) and presence (open circles) of 0.05 M Mg(ClO₄)₂ in phosphate buffer (0.05 M, pH 6.5).

In conclusion, the solubilization of DPPH^• in water by β-CD enables us to use the same ROS model radical, DPPH^•, both in aqueous and non-aqueous media and compare the reactivity of AscH₂ under the various reaction environments for the first time in this study. The DPPH^•-scavenging mechanism is largely affected by the reaction environments, such as solvents, pH, the presence of metal ions.

Author Contributions

Concept and design, IN; analysis and interpretation of data, IN, KO, and YS; drafting of the manuscript, IN; acquisition of data, YS; critical revision of the manuscript for important intellectual content, SF; study supervision, KF, TO, KM, and SF. All authors have read and agreed to the submitted version of the manuscript.

Abbreviations

AscH₂

ascorbic acid

β-CD

β-cyclodextrin

CV

cyclic voltammetry

DPPH^•

2,2-diphenyl-1-picrylhydrazyl radical

ET

electron transfer

HAT

hydrogen-atom transfer

iAscH₂

5,6-isopropylidene-l-ascorbic acid

KIE

kinetic isotope effect

MeCN

acetonitrile

MeOH

methanol

NaAscH

sodium l-ascorbate

PB

phosphate buffer

PT

proton transfer

PTIO^•

2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide

ROS

reactive oxygen species

SPLET

sequential proton-loss electron transfer

Conflict of Interest

No potential conflicts of interest were disclosed.