Kinetic Study of the Scavenging Reaction of the Aroxyl Radical by

Nov 13, 2014 - Recently, a new assay method that can quantify the aroxyl radical (ArO•) absorption capacity (ARAC) of antioxidants (AOHs) was propos...
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Kinetic Study of the Scavenging Reaction of the Aroxyl Radical by Seven Kinds of Rice Bran Extracts in Ethanol Solution. Development of an Aroxyl Radical Absorption Capacity (ARAC) Assay Method Kazuo Mukai,*,† Aya Ouchi,† Takumi Abe,‡ Kazumasa Murata,§ Kiyotaka Nakagawa,‡ and Teruo Miyazawa‡ †

Department of Chemistry, Faculty of Science, Ehime University, Matsuyama 790-8577, Japan Food and Biodynamic Chemistry Laboratory, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan § Agricultural Research Institute, Toyama Prefectural Agricultural, Forestry and Fisheries Research Center, Toyama 939-8153, Japan ‡

S Supporting Information *

ABSTRACT: Recently, a new assay method that can quantify the aroxyl radical (ArO•) absorption capacity (ARAC) of antioxidants (AOHs) was proposed. In the present work, the second-order rate constants (ksExtract) and ARAC values for the reaction of ArO• with seven kinds of rice bran extracts 1−7, which contain different concentrations of α-, β-, γ-, and δ-tocopherols and -tocotrienols (α-, β-, γ-, and δ-Tocs and -Toc-3s) and γ-oryzanol, were measured in ethanol at 25 °C using stopped-flow spectrophotometry. The ksExtract value (1.26 × 10−2 M−1 s−1) of Nipponbare (extract 1) with the highest activity was 1.5 times larger than that (8.29 × 10−3) of Milyang-23 (extract 7) with the lowest activity. The concentrations (in mg/100 g) of α-, β-, γ-, and δ-Tocs and -Toc-3s and γ-oryzanol found in the seven extracts 1−7 were determined using HPLC-MS/MS and UV−vis absorption spectroscopy, respectively. From the results, it has been clarified that the ArO•-scavenging rates (ksExtract) (that is, the relative ARAC value) obtained for the seven extracts 1−7 may be approximately explained as the sum of the product {Σ ksAOH‑i [AOH-i]/105} of the rate constant (ksAOH‑i) and the concentration ([AOH-i]/105) of AOH-i (Tocs, Toc-3s, and γ-oryzanol) included in rice bran extracts. The contribution of γ-oryzanol to the ksExtract value was estimated to be between 3.0−4.7% for each extract. Taken together, these results suggest that the ARAC assay method is applicable to general food extracts. KEYWORDS: rice bran extract, tocopherol, tocotrienol, free radicals, vitamin E, antioxidant activity, reaction rate, stopped-flow spectrophotometry, ARAC value, kinetic study

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ks AOH

INTRODUCTION In recent years, α-, β-, γ-, and δ-tocotrienols (α-, β-, γ-, and δ-Toc-3s) (Figure 1) have received much attention,1 because beneficial biological properties such as cholesterol lowering,2−4 anticancer,5,6 anti-inflammatory,7,8 antiangiogenic,9,10 and neuroprotective11 effects have been reported for Toc-3s. These findings suggest that Toc-3s have a wide variety of health benefits. Generally, the amounts of α-, β-, γ-, and δ-Toc-3s found in foods are much less than those of α-, β-, γ-, and δ-tocopherols (α-, β-, γ-, and δ-Tocs).12 However, rice bran and palm oil include comparatively high concentrations of Toc-3s and are used as the food source of Toc-3s.13−15 In recent years, various methods to assess the free radical scavenging activity of natural antioxidants and food extracts have been proposed.16 However, a kinetic study to assess the total scavenging activity of free radicals by antioxidants included in food extracts has not been performed. Previously, a kinetic study of the scavenging reaction of 2,6-di-t-butyl-4-(4methoxyphenyl)phenoxyl (abbreviated as aroxyl (ArO•)) radical (Figure 1) with natural antioxidants (AOHs), including α-, β-, γ-, and δ-Tocs and four tocopherol derivatives, was performed in ethanol solution at 25 °C.17 The second-order rate constants (ksAOH) for the reaction of AOHs with ArO• were measured using stopped-flow spectrophotometry (reaction 1). © 2014 American Chemical Society

ArO • + AOH ⎯⎯⎯⎯⎯→ ArOH + AO •

(1)

The second-order rate constants (ksAOH (f) and ksAOH (t1/2)) were determined by analyzing the first-order rate constant ( f) and the half-life (t1/2) of the decay curve of ArO•, respectively, and were found to be in good agreement with each other. From the results, a new assay method which can quantify the aroxyl radical absorption capacity (ARAC) of phenolic AOHs was proposed.17 It is a merit of ARAC assay method that ARAC value obtained is directly connected to the free radical scavenging rate (ksAOH (f) and ksAOH (t1/2)), as details are described in Result section. As rice is the staple food of many Asian peoples, including Japanese peoples, species improvement is often practiced, and many kinds of rice are cultivated.15 Rice brans include many kinds of AOHs, such as vitamin E homologues (4 Tocs and 4 Toc-3s), γ-oryzanol (γ-Ory) (that is, 10 kinds of phytosterols, including cycloartenyl ferulate) (Figure 1), polyphenols, and carotenoids.14,15,18−23 As reported in a previous study,15 rice Received: Revised: Accepted: Published: 11901

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brans, and to demonstrate that the ARAC method is applicable to not only pure α-, β-, γ-, δ-TocHs, but also general food extracts. Measurement of the rate constants (ksγ‑Ory (f) and ksγ‑Ory (t1/2)) for γ-Ory was also performed, as rice brans generally include high concentrations of γ-Ory.14,18,19,22 In addition, the concentrations (in mg/100 g) of α-, β-, γ-, δ-Tocs and -Toc-3s included in extracts 1−7 were determined using HPLC-MS/MS.24 The concentration of γ-Ory was determined from the UV−vis absorption spectra of rice bran extracts 1−7.14 Furthermore, comparison of the ksExtract (f) values observed for the above extracts 1−7 with the sum of the product {∑ ksAOH‑i (f) [AOH-i]/105} of the ksAOH‑i ( f) values obtained for each AOH-i and the concentration ([AOH-i]/105) of AOH-i included in the extracts was performed, in order to ascertain the validity of the ARAC assay method developed.17

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MATERIALS AND METHODS

Materials. α-, β-, γ-, δ-Tocs, and -Toc-3s were kindly supplied by Eisai Co. Ltd. (Tokyo, Japan). γ-Ory extracted from rice germ is commercially available (Wako Chemicals, Japan). The ArO• radical was prepared by following the method of Rieker et al.25 Preparation of Rice Bran Extracts 1−7. Seven kinds of crude rice bran samples 1−7 (Nipponbare (1), Wataribune (2), Koshihikari (3), Oiran (4), Moritawase (5), γ-rich Koshihikari (6), Milyang-23 (7); Table 1) were prepared from domestic Japanese (or Korean) dehulled rice using a Toyo-Tester grain-polishing machine (Toyo Rice Corporation, Tokyo, Japan).15 γ-Rich Koshihikari (extract 6), which includes high concentrations of γ-Toc and -Toc-3 (Table 2), is a mutant of Koshihikari (extract 3). The weight of each sample was approximately 10% of the dehulled rice. Measurements of the amounts of Tocs and Toc-3s and total γ-oryzanol in the rice bran were performed by Chen and Bergman14 and Sookwong et al.,15 using 2-propanol as an extraction solvent. In the present work, 2-propanol was similarly used to prepare the seven rice bran extracts 1−7. Specifically, freshly prepared rice bran powder (40.2 g) of Nipponbare (1) was ground in a mortar using a pestle and subsequently freeze-dried for 6 h. To 38.3 g of dried powder, 150 mL of 2-propanol was added, and extraction was performed for 10 min at 20 °C using a magnetic stirrer. The extraction was repeated 3 times. Approximately 400 mL of the extracted 2-propanol solution was evaporated using a rotary evaporator. The resultant oil was then dried for 2 h using a vacuum pump to eliminate the remaining 2-propanol and water. Finally, 7.75 g of rice bran extract 1 was obtained. Repeating the extraction by 2-propanol from dried powder resulted in a notable decrease in the weight of the extract, so that the final quantity of the extract obtained by the third extraction was only 8% of that of the first extraction. Rice bran extracts 2−7 were prepared similarly from ∼40 g of rice brans 2−7. An approximately 5% weight loss was observed by freezedrying rice brans 2−7 for 6 h. The weight ratios of rice bran extracts 1−7 to freeze-dried rice bran powders 1−7 were 0.200, 0.243, 0.191, 0.123, 0.196, 0.178, and 0.189, respectively, all of which were similar to each other with the exception of Oiran (4). Taken together, these results suggest that the method of preparation of rice bran extracts 1−7 via 2-propanol extraction is reasonable. Measurements of the ArO•-radical scavenging rates (ksExtract( f) and ksExtract(t1/2)) and the concentrations of 4 Tocs and 4 Toc-3s included in extracts 1−7 were performed using the extracts prepared as described above. HPLC-MS/MS Measurement of the Concentrations of α-, β-, γ-, δ-Tocopherols and -Tocotrienols Included in Rice Bran Extracts 1−7. Concentrations of α-, β-, γ-, δ-Tocs and -Toc-3s included in rice bran extracts 1−7 were determined by using a HPLC-MS/MS technique. Approximately 10 mg of rice bran extracts whose weight was correctly measured was dissolved in 3.00 mL of n-hexane. This solution (50.0 μL) was further diluted with n-hexane to yield a final volume of 250 μL. Next, a portion (20.0 μL) of the prepared solution was subjected to

Figure 1. Molecular structures of α-, β-, γ-, and δ-tocopherols (Tocs) and α-, β-, γ-, and δ-tocotrienols (Toc-3s), γ-oryzanol, and aroxyl radical (ArO•).

brans prepared from many kinds of rice were found to contain different concentrations of α-, β-, γ-, δ-Tocs and -Toc-3s. However, to the best of our knowledge, a detailed kinetic study of the free radical scavenging activity of rice brans has not been carried out. In the present study, measurements of the rate constants (ksExtract ( f) and ksExtract (t1/2)) and the relative ARAC values were performed for seven kinds of rice bran extracts 1−7 (Table 1), which were prepared from Japanese and Korean rice varieties, in ethanol, to evaluate the free radical scavenging activity of rice 11902

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Table 1. ksExtract (f) and ksExtract (t1/2) Values and Relative ARAC (f) and ARAC (t1/2) Values rice bran extract

ksExtract ( f) (L g−1 s−1)

ARAC ( f) value

−2

−3

1.26 × 10 1.08 × 10−2 1.07 × 10−2 9.30 × 10−3 9.30 × 10−3 8.70 × 10−3 8.29 × 10−3 2.64 × 10−2 11.9

Nipponbare (1) Wataribune (2) Koshihikari (3) Oiran (4) Moritawase (5) γ-rich Koshihikari (6) Milyang-23 (7) γ-oryzanol α-Toc

1.06 × 10 9.08 × 10−4 8.99 × 10−4 7.82 × 10−4 7.82 × 10−4 7.31 × 10−4 6.96 × 10−4 2.22 × 10−3 1.00

ksExtract (t1/2) (L g−1 s−1)

ARAC (t1/2) value

1.19 × 10−2 1.10 × 10−2 1.08 × 10−2 9.74 × 10−3 9.07 × 10−3 8.53 × 10−3 8.45 × 10−3 2.67 × 10−2 11.8

1.01 × 10−3 9.32 × 10−4 9.15 × 10−4 8.25 × 10−4 7.69 × 10−4 7.23 × 10−4 7.16 × 10−4 2.26 × 10−3 1.00

Table 2. Contents of 8 Vitamin E Homologues (4 Tocs and Toc-3s) and γ-Oryzanol Included in Rice Bran Extracts 1−7 rice bran extract Nipponbare (1) Wataribune (2) Koshihikari (3) Oiran (4) Moritawase (5) γ-rich Koshihikari (6) Milyang-23 (7)

α-Toc (mg/100 g)a

β-Toc (mg/100 g)

γ-Toc (mg/100 g)

δ-Toc (mg/100 g)

α-Toc-3 (mg/100 g)

β-Toc-3 (mg/100 g)

γ-Toc-3 (mg/100 g)

δ-Toc-3 (mg/100 g)

Total Tocs and Toc3s (mg/100 g)

γ-Ory (mg/100 g)

50.4 ± 4.2b 43.8 ± 5.3 37.4 ± 3.3 60.4 ± 1.0 33.5 ± 1.6 9.6 ± 0.1

2.0 ± 0.1 3.1 ± 0.3 1.7 ± 0.2 3.3 ± 0.3 2.7 ± 0.2 0.2 ± 0.1

5.0 ± 0.5 13.1 ± 0.6 6.8 ± 0.6 2.6 ± 0.1 3.0 ± 0.3 43.4 ± 2.0

0.4 ± 0.1 2.0 ± 0.3 0.6 ± 0.1 0.7 ± 0.1 0.7 ± 0.3 3.0 ± 0.4

34.5 ± 4.5 20.8 ± 0.5 22.1 ± 0.8 20.3 ± 0.4 24.3 ± 2.4 3.3 ± 0.2

0.3 ± 0.1 0.3 ± 0.1 0.2 ± 0.1 0.3 ± 0.1 0.6 ± 0.1 0.0 ± 0.0

40.8 ± 2.4 47.5 ± 2.4 47.2 ± 2.5 23.4 ± 0.8 46.1 ± 5.5 84.8 ± 6.3

2.9 ± 0.2 5.2 ± 0.3 2.7 ± 0.1 1.4 ± 0.1 4.9 ± 0.1 3.4 ± 0.5

136.3 135.8 118.7 112.3 115.9 147.6

1560 1400 1420 1400 1970 1610

11.2 ± 0.1

0.8 ± 0.1

24.1 ± 1.8

1.8 ± 0.3

8.4 ± 1.0

0.0 ± 0.0

99.4 ± 8.5

5.7 ± 0.4

151.5

1530

mg/100 g shows the contents (mg) of 4 Tocs and 4 Toc-3s and γ-Ory included in 100 g of rice bran extracts 1−7. bValues are expressed as mean ± SD (n = 3).

a

HPLC-MS/MS measurement.15,24 Separation was performed at 40 °C using a silica column (ZORBAX Rx-SIL, 4.6 × 250 mm; Agilent, Palo Alto, CA). A mixture of hexane/1,4-dioxane/2-propanol (100:4:0.5) was used as the mobile phase at a flow rate of 1.0 mL/min. Tocs and Toc-3s were detected in atmospheric pressure chemical ionization mode (APCI). MS/MS parameters were optimized with Tocs and Toc-3s standards in APCI mode (positive). Tocs and Toc-3s were detected using multiple reaction monitoring (MRM) as follows: α-Toc, m/z 431.3 > m/z 165.1; β-Toc, m/z 417.3 > m/z 151.3; γ-Toc, m/z 417.3 > m/z 151.0; δ-Toc, m/z 403.3 > m/z137.0; α-Toc-3, m/z 425.3 > m/z 165.1; β-Toc-3, m/z 411.3 > m/z 151.1; γ-Toc-3, m/z 411.3 > m/z 151.2; δ-Toc-3, m/z 397.2 > m/z 137.0. Typical MRM chromatograms of standard vitamin E homologues (A) and a rice bran extract (B) are shown in Figure S1A and B, respectively (Supporting Information). Tocs and Toc-3s concentrations in the rice bran extracts were calculated using calibration curves of standard Tocs and Toc-3s. The concentration was determined three times for each sample, and the average values are summarized in Table 2. UV−Vis Absorption Measurement of the Concentration of γ-Oryzanol Included in Rice Bran Extracts 1−7. As reported previously,14,19 γ-Ory extracted from rice germ oil contains 10 kinds of phytosterols. However, measurement of the concentration of each phytosterol included in rice bran extracts 1−7 was not performed in the present work, because the ArO•-scavenging rate of γ-Ory is two to 3 orders of magnitude smaller than those of α-, β-, γ-, and δ-Tocs and -Toc-3s, as will be discussed later. In the present work, the total concentration of γ-Ory included in rice bran extracts 1−7 was determined in ethanol from the UV−vis absorption spectrum, following the method performed by Chen and Bergman.14 The values obtained are listed in Table 2, together with those for α-, β-, γ-, and δ-Toc and -Toc-3. Measurements. Measurement of the second-order rate constants (ksAOH( f) and ksAOH(t1/2)) and ARAC values for the reaction of ArO• with AOH (reaction 1) was performed using a Unisoku single-mixing stopped-flow spectrophotometer (Model RSP-1000), by mixing equal volumes of ethanol solutions of ArO• and AOH under nitrogen atmosphere.17,26−28 The reaction was monitored with either single wavelength detection (Figure 2B) or a photodiode array detector (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(f) and ksAOH(t1/2)) were estimated to be about 7% in ethanol solution. Details of the measurements were described previously.17,26−28

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RESULTS AND DISCUSSION Rates of the Aroxyl-Radical-Scavenging (ks) of Rice Bran Extracts 1−7 and γ-Oryzanol in Ethanol Solution. UV−vis absorption spectra of rice bran extracts 1−7 and γ-Ory were measured in ethanol. The spectra observed for Koshihikari (3) and γ-Ory are shown in Figure 3A and B, respectively. The spectrum of Koshihikari (3) shows an absorption maximum (λmax) at 328 nm and a shoulder (λshoulder) at ∼300 nm. Similar spectra were observed for extracts 1, 2, and 4−7. The absorption at 328 nm is attributable to γ-Ory (λmax = 328 nm (εmax = 35.6 L g−1cm−1) and λshoulder ∼ 300 nm).14,19 Weak broad absorption in the region between 380−500 nm (Figure 3A) is due to several carotenoids included in rice brans, as previously reported.19 α-, β-, γ-, δ-Tocs and -Toc-3s show absorption maxima, λmax, between 292−298 nm in ethanol, as previously reported.29 The absorption bands of these Tocs and Toc-3s are thought to overlap with that of γ-Ory, because the εmax values (7.24−9.00 L g−1cm−1) of Tocs and Toc-3s are several times smaller than that of γ-Ory (εmax = 35.6 L g−1cm−1), and the concentrations of Tocs and Toc-3s are lower than that of γ-Ory (Table 2), as will be discussed later.29 The rate constant (ksExtract) for the reaction of the ArO• radical with rice bran extracts 1−7 was determined in ethanol at 25.0 °C (reaction 1). By reacting Oiran 4 with ArO• radical, the absorbance at 376 and 580 nm of the ArO• decreases, and the absorbance at 428 nm of Toc• and Toc-3• radicals, which will be mainly due to α-Tocs• and α-Toc-3•, increases, as shown in Figure 2A.27,28 The decay rate (kobsd( f)) of the ArO• radical was measured by following the decrease in absorbance at 580 nm (Figure 2B), because the absorption at 376 nm overlaps with that of Oiran 4 (specifically that of γ-Ory, as described above19). The pseudo-first-order rate constants (kobsd(f)) at 580 nm were 11903

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Figure 2. (A) Change in electronic absorption spectra of ArO• and Toc• (and Toc-3•) radicals during the reaction of ArO• with Oiran (4) in ethanol solution at 25.0 °C. The initial concentrations are [ArO•] = 1.14 × 10−4 M and [Oiran] = 98.1 g/L. The spectra were recorded at 1 s intervals. The arrow indicates a decrease (ArO•) and increase (Toc• and Toc-3•) in absorbance with time. (B) Time dependences of the absorbance of the ArO• radical (at 580 nm) in solution containing five different concentrations of extract 4 at 25.0 °C. (C) Pseudo-first-order rate constant (kobsd (f)) versus [Oiran] plot. (D) Baseline correction. (E) ln 2/t1/2 versus [Oiran] plot.

were obtained by plotting kobsd(f) against [Extract], as shown in Figure 2C. Units of g/L were used for the concentrations of rice bran extracts 1−7. The ks(f) value obtained for Oiran (4) is 9.30 × 10−3 L g−1s−1 (Table 1). Equation 3 is easily obtained by substituting the relation for the first-order reaction (kobsd( f) = ln 2/t1/2) into eq 2:

linearly dependent on the concentration of the extract ([Extract]), and thus the rate equation is expressed as −d[ArO•]/dt = kobsd(f ) [ArO•] = ks Extract(f ) [Extract] [ArO•]

(2)

where ks (f) is the second-order rate constant for the oxidation of the extract by the ArO• radical. The rate constants (ks(f))

ln 2/t1/2 = ks Extract(t1/2) [Extract] 11904

(3)

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Figure 3. UV−vis absorption spectra of (A) Koshihikari (extract 3) and (B) γ-oryzanol in ethanol.

where t1/2 is the half-life of ArO• in the presence of extract. To obtain the correct t1/2 value in eq 3, baseline correction was performed for the decay curve of ArO• in Figure 2B. The results obtained are shown in Figure 2D. Equation 3 indicates that the ksExtract (t1/2) value can be obtained from a plot of ln 2/t1/2 versus [Extract]. An example for Oiran (4) is shown in Figure 2E. The value of ksOiran(t1/2) (= 9.74 × 10−3 L g−1s−1) obtained for Oiran (4) was in good agreement with that of ksOiran(f) (= 9.30 × 10−3 L g−1s−1), as listed in Table 1. Similar measurements were performed for the reaction of ArO• with extracts 1−3, 5−7, and γ-Ory in ethanol solution. The result obtained for γ-Ory is shown in Figure 4. The ksExtract( f) and ksExtract(t1/2) values obtained for extracts 1−7 are summarized in Table 1, together with those of γ-Ory and α-Toc. Both ksExtract( f) and ksExtract(t1/2) increase in the following order:

Figure 4. (A) Change in electronic absorption spectrum of the ArO• radical during the reaction of ArO• with γ-Ory in ethanol at 25.0 °C. [ArO•] = 6.50 × 10−5 M and [γ-Ory] = 2.96 g/L. (B) Time dependences of the absorbance of the ArO• radical (at 580 nm) in ethanol containing eight different concentrations of γ-Ory at 25.0 °C. (C) Pseudo-first-order rate constant (kobsd(f)) versus [γ-Ory] plot.

Milyang‐23 (7) ≤ γ ‐rich Koshihikari (6) < Moritawase (5) ∼ Oiran (4) < Wataribune (3) ∼ Koshihikari (2) < Nipponbare (1) < γ ‐oryzanol < α ‐Toc

(4)

proposed previously,17 relative ARAC (f) and ARAC (t1/2) values for AOH were defined according to eqs 5 and 6:

Extract

Nipponbare (1) exhibits a ks ( f) value 1.5 times larger than that of Milyang-23 (7). The ksγ‑Ory(f) value of γ-Ory is 2.1 times larger than that of Nipponbare (1). However, the value is 450 times smaller than that of α-Toc. The ksExtract(f) values obtained for extracts 1−7 are shown as a bar graph in Figure 5A. Development of an Aroxyl Radical Absorption Capacity (ARAC) Assay Method for Food Extracts. As

relative ARAC (f ) value (based on molar concentration unit (M = mol/L)) = ks AOH(f ) (M−1 s−1) /ks α‐Toc(f ) (M−1 s−1) 11905

(5)

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eqs 5 and 6. The relative ARAC ( f) (and ARAC (t1/2)) values obtained for extracts 1−7 are shown as a bar graph in Figure 5B. As summarized in Table 1, the ksExtract(f) and ksExtract(t1/2) values and the relative ARAC (f) and ARAC (t1/2) values obtained for rice bran extracts 1−7 and γ-Ory are in good agreement with each other, suggesting that the ARAC assay method developed is applicable to the estimation of the free radical scavenging activity of food extracts. Measurement of ARAC values for many kinds of food extracts, including vegetable and fruit extracts and edible oils, will be necessary to verify the applicability of the ARAC assay method to food extracts, as performed for the SOAC assay method.29,36−38 Contents of Eight Vitamin E Homologues and γ-Oryzanol in Rice Bran Extracts 1−7. To compare the ksExtract(f) value observed for rice bran extracts 1−7 with the value of ksExtract(f) calculated using the rate constants (ksAOH‑i(f)) and the concentrations ([AOH-i]) of each AOH included in extracts 1−7, measurements of the concentrations of 4 Tocs and 4 Toc-3s and γ-Ory were performed. The concentrations (in mg/100 g) of 4 Tocs and 4 Toc-3s included in extracts 1−7 were measured using HPLC-MS/MS, as described in Materials and Methods. The results obtained are listed in Table 2. The concentrations of α-, β-, γ-, δ-Tocs, and -Toc-3s obtained for extracts 1−7 are in reasonable agreement with previously reported values.15 High concentrations of Toc-3s are included in rice bran extracts 1−7, as listed in Table 2. Nipponbare (1) and Oiran (4) contain high total concentrations of α-Toc and α-Toc-3. Specifically, high concentrations of γ-Toc and γ-Toc-3 were included in γ-rich Koshihikari (6) and Milyang-23 (7). On the other hand, the total concentration of γ-Ory included in extracts 1−7 was determined from the UV−vis absorption spectrum. As reported previously,14,19 γ-Ory extracted from rice germ oil contains 10 kinds of phytosterols. Three of these, namely, cycloartenyl ferulate, 24-methylenecycloartanyl ferulate, and campesteryl ferulate, were major components of γ-Ory. In the present work, commercially available γ-Ory extracted from rice germ oil was used as a standard. The εmax value (35.6 L g−1cm−1) of γ-Ory at 328 nm in ethanol was determined from the UV−vis absorption spectrum of γ-Ory (see Figure 3B). Using this εmax value, the concentration (in mg/100 g) of γ-Ory included in extracts 1−7 was tentatively estimated. The values obtained are listed in Table 2, together with those for α-, β-, γ-, and δ-Toc, and -Toc-3. The concentration of γ-Ory in extracts 1−7 were similar (between 1400−1970 mg/100 g), and about 1 order of magnitude larger than those of total Tocs and Toc-3s (112−152 mg/100 g), as listed in Table 2. Comparison of Observed and Calculated ArO•Scavenging Activities for Rice Bran Extracts 1−7. It is well-known that various AOHs coexist not only in foods and plants, but also in human tissues. The ksExtract(f) (L g−1s−1) values for rice bran extracts 1−7 were determined from a plot of kobsd( f) versus [Extract (g/L)] using eq 7.

Figure 5. Comparison of (A) the ks( f) values and (B) the relative ARAC (t1/2) values for seven kinds of rice bran extracts 1−7 in ethanol.

relative ARAC (t1/2) value = ks AOH(t1/2) (M−1 s−1) /ks α‐Toc(t1/2) (M−1 s−1) = (t1/2 α‐Toc/t1/2 AOH) × ([α ‐Toc]/[AOH])

(6)

Equation 5 indicates that the ARAC (f) value corresponds to the ratio (ksAOH( f)/ksα‑Toc( f)) of the scavenging rate of ArO• (ksAOH(f)) by AOH to that (ksα‑Toc(f)) by α-Toc. α-Toc was used as a standard compound of ARAC assay. By substituting the relation eq 3 into the second line of eq 6, we can obtain the third line of eq 6. According to eq 6, ARAC (t1/2) value may be determined by the measurement of the half-life of the ArO• radical (t1/2AOH) and the concentration of AOH ([AOH]) without analysis of the decay rate of ArO•. In the ARAC assay method, α-Toc was used as a standard compound for the following reasons: (i) α-Toc is a representative phenolic antioxidant, (ii) α-Toc shows the same order of ks value as those for the other phenolic antioxidants (such as tocopherol homologues (4 Tocs and 4 Toc-3s), catechins, and ubiquinol10),17,26−28,30 (iii) α-Toc and the above phenolic antioxidants often coexist in general foods31−33 and biological systems (such as plasma34 and mitochondria35), and function as free radical scavengers at a similar reaction field. Furthermore, α-Toc was used as a standard compound in the singlet oxygen absorption capacity (SOAC) assay method, which was recently developed to evaluate the singlet oxygen quenching activity of many natural AOHs and food extracts.36−38 Units of g/L were used for the concentrations of rice bran extracts 1−7, while units of L g−1 s−1 were used for ksExtract(f) and ksExtract(t1/2) values. The ARAC values for extracts 1−7 were also calculated using g/L as the unit of concentration for AOH in

kobsd(f ) = ks Extract(f ) [Extract]

(7)

kobsd(f ) = {Σks AOH‐i(f )[AOH‐i]/105} [Extract]

(8)

As listed in Table 2, 4 Tocs and 4 Toc-3s and γ-Ory (abbreviated as AOH-i) are included in extracts 1−7. Polyphenols are also included in rice bran.21,23 However, the concentrations of these polyphenols are not reported. Furthermore, carotenoids are included in rice bran extracts, as shown in Figure 3A.21 However, as the free radical scavenging activity of 11906

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Table 3. Comparison between Observed and Calculated ArO• Scavenging Rates (ksKoshihikari (Obsd.) and ΣksAOH‑i (f) × ([AOH-i]/105) (Calcd.)) for Koshihikari (3) in Ethanol Solution antioxidant

ksAOH‑i( f) (M−1 s−1)

Mw

ksAOH‑i( f) (L g−1 s−1)

[AOH-i] (mg/100 g)

ksAOH‑i( f) × ([AOH-i]/105) (L g−1 s−1)

α-Toc α-Toc-3 β-Toc β-Toc-3 γ-Toc γ-Toc-3 δ-Toc δ-Toc-3 γ-Oryzanol Σ ksAOH‑i( f) × ([AOH-i]/105) (Calcd.) ksExtract( f) (Obsd.)

5.12 × 10 4.97 × 103 2.77 × 103 3.11 × 103 2.82 × 103 2.76 × 103 1.02 × 103 1.09 × 103

430.71 424.66 416.69 410.64 416.69 410.64 402.67 396.62

11.9 11.7 6.65 7.57 6.77 6.72 2.53 2.75 2.64 × 10−2

37.4 22.1 1.7 0.2 6.8 47.2 0.6 2.7 1420

4.45 × 10−3 2.58 × 10−3 0.115 × 10−3 0.0126 × 10−3 0.460 × 10−3 3.17 × 10−3 0.0152 × 10−3 0.0743 × 10−3 0.375 × 10−3 11.3 × 10−3

3

10.7 × 10−3

Table 4. Comparison between Observed and Calculated ArO•-Scavenging Rates (ksExtract( f) (Obsd.) and ksExtract(f) (Calcd.)) for Rice Bran Extracts 1−7 in Ethanol, Ratio of the Rate Constants (ksExtract( f) (Obsd.)/ksExtract( f) (Calcd.)), and Contribution of γ-Oryzanol (ksγ‑Ory( f) (Calcd.)) rice bran extract

ksExtract( f) (Obsd.) (L g−1 s−1)

ksExtract( f) (Calcd) (L g−1 s−1)

ksExtract( f) (Obsd.)/ksExtract( f) (Calcd)

ksγ‑Ory( f) (Calcd) (contribution of γ-Ory)

Nipponbare (1) Wataribune (2) Koshihikari (3) Oiran (4) Moritawase (5) γ-rich Koshihikari (6) Milyang-23 (7)

1.26 × 10−2 1.08 × 10−2 1.07 × 10−2 9.30 × 10−3 9.30 × 10−3 8.70 × 10−3 8.29 × 10−3

1.38 × 10−2 1.25 × 10−2 1.13 × 10−2 1.20 × 10−2 1.10 × 10−2 1.08 × 10−2 1.13 × 10−2

0.913 0.864 0.947 0.775 0.845 0.806 0.734

4.12 × 10−4 3.70 × 10−4 3.75 × 10−4 3.70 × 10−4 5.20 × 10−4 4.25 × 10−4 4.04 × 10−4

corresponding (ksExtract(f) (Calcd.)) values calculated using only the concentrations of 4 Tocs, 4 Toc-3s, and γ-Ory. We may expect that the ksExtract(f) (Obsd.) values for extracts 1−7 are similar to or larger than those (ksExtract(f) (Calcd.)) calculated, because the other AOHs, such as polyphenols included in extracts 1−7, may also contribute to the ksExtract( f) (Obsd.) values.26 In a previous study, measurements of the ArO•-scavenging rate constants (ksAOH( f)) of AOHs (α-Toc, ubiquinol-10 (UQ10H2) and sodium ascorbate (Na+AsH−)) were performed in 2-propanol/water (5/1, v/v) solution.28 ksAOH( f) values were measured not only for each AOH but also for the mixtures of two AOHs ((i) α-Toc and UQ10H2 and (ii) α-Toc and Na+AsH−). A notable synergistic effect, namely, that the ksAOH(f) values increase 1.6, 2.5, and 6.8 times for α-Toc, UQ10H2, and Na+AsH−, respectively, was observed for the solutions containing two kinds of AOHs. Furthermore, measurements of the ArO•-scavenging rate constants (ksAOH( f)) of AOHs (α-, β-, γ-, δ-Tocs) were recently performed in ethanol solution, not only for each AOH but also for the mixtures of two AOHs ((i) α-Toc and β-Toc, (ii) α-Toc and γ-Toc, and (iii) α-Toc and δ-Toc).30 A considerable synergistic effect, namely, that the ksAOH( f) value increases 12%, was observed for the solutions containing α-Toc and γ-Toc. On the other hand, a canceling effect, namely that the ksAOH( f) value decreases by (i) 7% and (ii) 24%, respectively, was observed for the solutions containing (i) α-Toc and β-Toc and (ii) α-Toc and δ-Toc. Similar results were observed for the mixtures of α-, β-, γ-, δ-Toc-3s.30 In rice bran extracts 1−3 and 5, the concentrations of total α-Toc and α-Toc-3 and of total γ-Toc and γ-Toc-3 were higher than those of total β-Toc and β-Toc-3 (and total δ-Toc and δ-Toc-3), while the concentrations of total α-Toc and α-Toc-3

carotenoids is generally low, the contribution of carotenoids to the ArO•-scavenging rate is thought to be small and negligible. Recently, measurements of the ArO•-scavenging rate constants (ksAOH(f)) of AOHs (α-, β-, γ-, δ-Tocs and -Toc-3s) were performed in ethanol solution.30 ksAOH(f) values of α-, β-, γ-, δ-Toc-3s were in good agreement with those of the corresponding α-, β-, γ-, δ-Tocs. The ksAOH( f) values reported are listed in Table 3. The ksAOH( f) value for γ-Ory was measured in the present work. Consequently, the ksExtract(f) values for extracts 1−7 were calculated using eq 8, that is, {Σ ksAOH‑i(f) [AOH-i]/105}, where the ksAOH‑i( f) (L g−1s−1) values are the rate constants for 4 Tocs, 4 Toc-3s, and γ-Ory in ethanol (see Tables 3 and 4). [AOH-i] is the concentration (in mg/100 g) of Tocs, Toc-3s, and γ-Ory included in extracts 1−7 (see Table 2). As an example, the result of the detailed analysis performed for Koshihikari (3) is listed in Table 3. Good agreement between the observed ksKoshihikari( f) value (= 1.07 × 10−2 L g−1s−1) and calculated {Σ ksAOH‑i( f) [AOH-i]/105} value (= 1.13 × 10−2 L g−1s−1) observed for Koshihikari (3). The contribution of γ-Ory to the total ksKoshihikari( f) value is small and about 3.3% of the total rate. A similar result was obtained for Nipponbare (1) (see Table 4). These results indicate that the total ArO•-scavenging activity of Nipponbare (1) and Koshihikari (3) may be explained by only considering the contribution of 8 kinds of vitamin E homologues and γ-Ory included in Nipponbare (1) and Koshihikari (3). The contribution of the other AOHs is negligible. Furthermore, this result suggests that the interactions between 8 vitamin E homologues and among vitamin E homologues and many molecules included in rice bran extracts are weak and negligible in solution. On the other hand, as listed in Table 4, ksExtract(f) (Obsd.) values for extracts 2 and 4−7 are 14−27% smaller than the 11907

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were similar to those of total γ-Toc and γ-Toc-3 (Table 2). Consequently, we may expect a synergistic effect (that is, an increase of the ksExtract(f) values) for rice bran extracts 1−3 and 5. However, the ksExtract(f) values observed for extracts 1−3 and 5 were similar to or smaller than the corresponding calculated values. High concentrations of proteins, lipids, and carbohydrates are included in rice bran extracts. The interactions between these compounds and Tocs (and Toc-3s) molecules may affect the ArO•-radical scavenging activity of AOHs. However, the reason is not clear at present. A detailed study will be necessary to clarify the reason why the values of the ratio (ksExtract(f) (Obsd.)/ ksExtract( f) (Calcd.)) obtained for extracts 1−7 are ≤1 (Table 4). In recent years, various methods to assess the free radical scavenging activity of natural antioxidants and vegetable and fruit extracts have been proposed.16 The oxygen radical absorbance capacity (ORAC) assay method is one of the most frequently used methods.39−42 The ORAC values for extracts of nearly 300 vegetables and fruits have been measured and are listed on the US Department of Agriculture (USDA) Web site (http://www. ars.usda.gov/SP2UserFiles/Place/12354500/Data/ORAC/ ORAC07.pdf). However, to our regret, in 2012 the USDA’s Nutrient Data Laboratory (NDL) removed the USDA ORAC Database for Selected Foods from the NDL Web site because of mounting evidence that the values indicating antioxidant capacity have no relevance to the effects of specific bioactive compounds, including polyphenols, on human health.43 Accordingly, several alternative methods have been proposed to assess antioxidant capacity against free radicals.16,44 In a previous work, a new assay method that can quantify the aroxyl radical absorption capacity (ARAC) of phenolic AOHs, including α-, β-, γ-, δ-Tocs and five tocopherol derivatives, was proposed.17 However, the examples of food extracts investigated were limited to a palm oil extract. In the present work, relative ARAC ( f) and ARAC (t1/2) values were measured for seven rice bran extracts 1−7, which include eight kinds of vitamin E homologues (4 Tocs and 4 Toc-3s) and γ-Ory. Results suggest that the ARAC assay method may be applicable not only to general natural AOHs but also to food extracts that include many kinds of AOHs.

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ASSOCIATED CONTENT

S Supporting Information *

Figure showing typical MRM chromatograms of standard vitamin E homologues (A) and a rice bran extract (B). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: 81-89-927-9588. Fax: 81-89-927-9590. E-mail: mukai-k@ dpc.ehime-u.ac.jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are very grateful to Prof. Shin-ichi Nagaoka of Ehime University for his kind support. We also are very grateful to Prof. Hisako Sato and to Eri Ishikawa of Ehime University for their kind help in the preparation of rice bran extracts. 11908

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