Isolation and Characterization of the 2, 2′-Azinobis (3

Sep 8, 2016 - bilberry,2 strawberry tree,3 pear,4,5 and marjoram.6 Arbutin is known to inhibit tyrosinase, a key enzyme in melanin synthesis, and it i...
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Isolation and Characterization of the 2,2′-Azinobis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS) Radical CationScavenging Reaction Products of Arbutin Akihiro Tai,*,† Asako Ohno,† and Hideyuki Ito‡ †

Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, Shobara, Hiroshima 727-0023, Japan Faculty of Health and Welfare Science, Okayama Prefectural University, Soja, Okayama 719-1197, Japan

J. Agric. Food Chem. 2016.64:7285-7290. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/09/19. For personal use only.

‡

ABSTRACT: Arbutin, a glucoside of hydroquinone, has shown strong 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation-scavenging activity, especially in reaction stoichiometry. This study investigated the reaction mechanism of arbutin against ABTS radical cation that caused high stoichiometry of arbutin in an ABTS radical cation-scavenging assay. HPLC analysis of the reaction mixture of arbutin and ABTS radical cation indicated the existence of two reaction products. The two reaction products were purified and identified to be a covalent adduct of arbutin with an ABTS degradation fragment and 3-ethyl6-sulfonate benzothiazolone. A time-course study of the radical-scavenging reactions of arbutin and the two reaction products suggested that one molecule of arbutin scavenges three ABTS radical cation molecules to generate an arbutin−ABTS fragment adduct as a final reaction product. The results suggest that one molecule of arbutin reduced two ABTS radical cation molecules to ABTS and then cleaved the third ABTS radical cation molecule to generate two products, an arbutin−ABTS fragment adduct and 3-ethyl-6-sulfonate benzothiazolone. KEYWORDS: arbutin, antioxidant, radical-scavenging activity, ABTS radical cation, adduct formation

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INTRODUCTION Arbutin, 1, a naturally occurring glucoside of hydroquinone (Figure 1), is found in diverse plants such as bearberry,1

antioxidant activities with attention to the above-mentioned proposals and viewpoints.17−23 We found that arbutin exhibited strong antioxidant activity comparable or superior to that of hydroquinone in a 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation-scavenging assay, an oxygen radical absorbance capacity assay, and two cell-based antioxidant assays.20 The amount of ABTS radical cation, 2 (Figure 1), scavenged by arbutin in the first minute was nearly the same as that scavenged by hydroquinone. Interestingly, the reaction of arbutin with ABTS radical cation proceeded until 30 min, and the reaction stoichiometry of arbutin was much higher than that of hydroquinone. In addition, the reaction solution of arbutin with ABTS radical cation changed to a purple color. Generally, the characteristic color of ABTS radical cation is discolored from dark green to light green when the radical is bleached by antioxidants. It has been reported that the reaction of an ABTS radical cation with an oxidation product of naringin results in the formation of a purple-colored compound24 and that indole-3-carbinol quenches ABTS radical cation to give a pink-colored covalent adduct with the radical.25 However, the structures of these colored compounds have not yet been elucidated. The high stoichiometry of arbutin in the ABTS radical cation-scavenging assay might be caused by the formation of a purple-colored compound. In this study, we investigated the reaction mechanism of arbutin against ABTS radical cation that caused high stoichiometry of arbutin in an ABTS radical cation-scavenging

Figure 1. Chemical structures of arbutin and ABTS radical cation.

bilberry,2 strawberry tree,3 pear,4,5 and marjoram.6 Arbutin is known to inhibit tyrosinase, a key enzyme in melanin synthesis, and it is used as a skin-lightening agent for cosmetic products.7 It has recently been reported that arbutin was effective for the treatment of postinflammatory hyperpigmentation, which is a reactive hypermelanosis and sequela of various inflammatory skin conditions,8 and that arbutin exhibited anti-inflammatory effects with down-regulation of inducible NO synthase and proinflammatory cytokines in lipopolysaccharide-stimulated BV2 microglial cells.9 Some studies have shown that arbutin had antioxidant activity but that the antioxidant activity was not as strong as that of hydroquinone.10−12 However, in those studies, the antioxidant activity of arbutin was evaluated mainly from the viewpoint of kinetics10 or in a relatively short period.11,12 It has been proposed that the reactivity of antioxidants should be investigated on the basis of reaction rate and reaction stoichiometry because there are two types of antioxidants scavenging radicals quickly and quenching many radicals and that multiple methods should be used to assess antioxidant activity because the activities of some antioxidants vary depending on the assay method.13−16 We have investigated © 2016 American Chemical Society

Received: Revised: Accepted: Published: 7285

June 27, 2016 September 4, 2016 September 8, 2016 September 8, 2016 DOI: 10.1021/acs.jafc.6b02847 J. Agric. Food Chem. 2016, 64, 7285−7290

Article

Journal of Agricultural and Food Chemistry

Purification and Structural Determination of the Reaction Products of Arbutin with ABTS Radical Cation. Arbutin (102.1 mg, 0.375 mmol) was dissolved in 750 mL of 100 mM citric acid− sodium citrate buffer (pH 6) and then mixed with 750 mL of 2.0 mM ABTS radical cation (1.5 mmol) in 10 mM citric acid−sodium citrate buffer (pH 4). The solution was reacted at 30 °C for 2 h. The reaction mixture was concentrated to about 350 mL and was then chromatographed on a 31.2 cm × 7.0 cm i.d. Diaion HP20 column (Mitsubishi Chemical, Tokyo, Japan) eluted with H2O and 60% methanol−H2O. The 60% methanol eluate was concentrated to dryness. The residue (1.01 g) was chromatographed on a 41.5 cm × 4.0 cm i.d. Toyopearl HW-40C column (Tosoh, Tokyo, Japan) eluted with 725 mL of 10% methanol−H2O to give 29 fractions. Fractions 9 and 10 were mixed and concentrated to give a purple powder (146.2 mg). Fractions 11−15 (102.8 mg) were chromatographed on a 26.0 cm × 1.0 cm i.d. Toyopearl HW-40F column (Tosoh) eluted with 50 mL of 10% methanol−H2O at a flow rate of 0.7 mL/min to give 10 fractions. Fractions 5 and 6 (61.9 mg) were again chromatographed on a 26.0 cm × 1.0 cm i.d. Toyopearl HW-40F column eluted with 40 mL of 10% methanol−H2O at a flow rate of 0.5 mL/min to give 20 fractions. Fractions 5−11 (51.3 mg) were further chromatographed on a 13.1 cm × 1.0 cm i.d. Wakogel 50NH2 column (Wako Pure Chemical Industries, Osaka, Japan) eluted with a stepwise gradient of acetone−H2O solvent system (100, 95, 90, 80, 60, 40, and 20%, v/v) to give 40 fractions. Fractions 20−33 were mixed and concentrated to give a white powder (48.7 mg). NMR spectra were obtained on a Varian NMR System 600 MHz instrument. Electron spray ionization (ESI) high-resolution mass spectra were recorded on a Bruker Daltonics MicrOTOF II instrument using direct sample injection. Purple powder (3): 1H NMR (600 MHz, CD3OD) δH 0.94 (3H, br t, J = 6.6 Hz, H-11), 3.23 (1H, m, H-5″), 3.29 (1H, br t, J = 7.8 Hz, H2″), 3.35 (1H, m, H-3″), 3.35 (1H, m, H-4″), 3.62 (1H, br d, J = 11.4 Hz, Ha-6″), 3.67 (1H, br d, J = 11.4 Hz, Hb-6″), 3.74 (2H, br d, J = 6.6 Hz, H-10), 4.41 (1H, br d, J = 6.6 Hz, H-1″), 5.24 (1H, br d, J = 9.6 Hz, H-6′), 6.02 (1H, br d, J = 9.6 Hz, H-5′), 6.09 (1H, br s, H-3′), 6.84 (1H, br d, J = 8.4 Hz, H-4), 7.23 (1H, br d, J = 8.4 Hz, H-5), 7.42 (1H, br s, H-7); 13C NMR (150 MHz, CD3OD) δC 13.3 (C-11), 42.6 (C-10), 61.1 (C-6″), 69.8 (C-4″), 73.5 (C-2″), 76.3 (C-3″), 77.1 (C5″), 99.0 (C-3′), 100.8 (C-1″), 112.9 (C-4), 122.1 (C-7), 126.1 (C-5), 126.3 (C-8), 129.3 (C-6′), 138.1 (C-5′), 139.7 (C-6), 141.4 (C-9), 147.2 (C-2′), 153.1 (C-4′), 174.7 (C-2), 183.5 (C-1′); ESI-HRMS m/ z [M − H]− calcd for C21H22N3O10S2 540.0752, found 540.0745. White powder (4): 1H NMR (600 MHz, CD3OD) δH 1.34 (3H, t, J = 7.5 Hz, H-11), 4.11 (2H, q, J = 7.5 Hz, H-10), 7.45 (1H, d, J = 8.4 Hz, H-4), 7.88 (1H, d, J = 8.4 Hz, H-5), 8.04 (1H, s, H-7); 13C NMR (150 MHz, CD3OD) δC 13.1 (C-11), 39.3 (C-10), 112.5 (C-4), 121.6 (C-7), 123.7 (C-5), 125.8 (C-8), 139.6 (C-6), 140.5 (C-9), 173.2 (C2); ESI-HRMS m/z [M − H]− calcd for C9H8NO4S2 257.9900, found 257.9906. Time Course of Reaction Product Formation and Arbutin Consumption. Arbutin (200 μM) was reacted with ABTS radical cation (1.0 mM) in 50 mM citric acid−sodium citrate buffer (pH 6). At the indicated time, an aliquot of the reaction mixture was withdrawn, and the concentrations of reaction products and arbutin were determined by HPLC. HPLC analysis was performed with a system consisting of LC-10AT pumps, an SPD-M10AVP diode array detector, a CTO-10A column oven, and a CBM-10A communications bus module. The chromatographic column was a 250 mm × 4.6 mm i.d., 5 μm, Inertsil Ph-3 column kept at 40 °C, and the mobile phase consisted of methanol/water/acetic acid (5:94:1, v/v/v) (solvent A) and methanol/water/acetic acid (30:69:1, v/v/v) (solvent B). A gradient was applied at a flow rate of 0.7 mL/min as follows: the proportion of solvent B in the eluent was increased from 0 to 100% (t = 30 min), kept at 100% (t = 35 min), and then decreased to 0% (35.1 min) until the next injection (t = 50 min). The absorbance at 280 and 550 nm was monitored. ABTS Radical Cation-Scavenging Assay. ABTS radical cationscavenging activities were assessed as described previously.26 Briefly, ABTS radical cation (100 μM) generated with an ABTS/hydrogen peroxide/peroxidase system was mixed with an antioxidant (20 or 60

assay. First, we performed a high-performance liquid chromatography (HPLC) analysis of the reaction mixture of arbutin and ABTS radical cation and identified reaction products of arbutin with ABTS radical cation. Then we carried out time-course studies of the radical-scavenging activity of reaction products and arbutin to determine both reaction rate and stoichiometry with ABTS radical cation, and we elucidated the radical-scavenging property of arbutin against ABTS radical cation.

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MATERIALS AND METHODS

Chemicals. ABTS diammonium salt and peroxidase from horseradish (type VI-A, essentially salt-free, 1310-units/mg solid) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Arbutin was obtained from Nacalai Tesque Inc. (Kyoto, Japan). Hydrogen peroxide solution (H2O2, 3%) was from Aldrich Chemical (Milwaukee, WI, USA). Reagents were used without further purification. All water used was of Milli-Q grade. Optimization of Reaction Conditions for Purple-Colored Product Formation. Arbutin (20 μM) was reacted with ABTS radical cation (100 μM) in 50 mM citric acid−sodium citrate buffer (pH 6), and arbutin (167, 200, 250, 333, and 500 μM) was reacted with ABTS radical cation (1.0 mM) in 50 mM citric acid−sodium citrate buffer (pH 6). ABTS radical cation was generated with an ABTS/hydrogen peroxide/peroxidase system as described previously.26 The mixture (1 mL) was kept at room temperature for 15 or 120 min. An aliquot of the reaction mixture was withdrawn and subjected to HPLC analysis. HPLC analysis was performed with a system consisting of LC-10AT pumps, an SPD-M10AVP diode array detector, a CTO-10A column oven, and a CBM-10A communications bus module (Shimadzu, Kyoto, Japan). The separation of arbutin and reaction products was achieved by isocratic elution on a 250 mm × 4.6 mm i.d., 5 μm, Inertsil Ph-3 column (GL Sciences Inc., Tokyo, Japan) thermostated at 40 °C with methanol/water/acetic acid (20:79:1, v/v/ v) at a flow rate of 0.7 mL/min. The absorbance at 280 nm was monitored for typical chromatograms of the reaction mixture presented in Figure 2. The absorbance at 280 nm for arbutin and at 550 nm for the reaction product at tR 28 min was also monitored.

Figure 2. HPLC chromatograms of the reaction between arbutin and ABTS radical cation: (A) arbutin (20 μM) reacted with ABTS radical cation (100 μM) for 120 min in sodium citrate buffer (50 mM, pH 6); (B) arbutin (200 μM) reacted with ABTS radical cation (1.0 mM) for 15 min in sodium citrate buffer (50 mM, pH 6). 7286

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Journal of Agricultural and Food Chemistry μM) in 50 mM citric acid−sodium citrate buffer (pH 6). The reaction was carried out at room temperature for 2 h. The decrease of ABTS radical cation concentration was monitored by measuring the absorbance at 730 nm with a U-1900 spectrophotometer (Hitachi, Tokyo, Japan). The number of ABTS radical cations scavenged by each antioxidant was calculated by using the equation RSA(n) = (ΔA120 /A 0) × [ABTS radical cation]/[antioxidant]

where RSA(n) = radical-scavenging activity factor n, the moles of radicals scavenged by each mol of antioxidant; ΔA120 = absorbance difference between the reaction solution and control at 120 min; A0 = initial absorbance of the control; [ABTS radical cation] = ABTS radical cation concentration; and [antioxidant] = antioxidant concentration.

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Figure 3. Effects of different ratios of the arbutin/ABTS radical cation on the formation of reaction product at tR 28 min and on the consumption of arbutin. Values are means ± SD of three separate experiments.

RESULTS AND DISCUSSION Hydroquinone is a potent antioxidant having two oxidizable hydroxyl groups on the benzene ring. Arbutin, a glucoside of hydroquinone (Figure 1), retains one of the oxidizable hydroxyl groups of hydroquinone and was supposed to have decreased antioxidant activity according to the classical structure−activity relationship theory. In fact, the 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity of arbutin has been shown in previous studies to be less than that of hydroquinone in reaction rate and reaction stoichiometry.11,12,20 However, arbutin exhibited strong ABTS radical cation-scavenging activity in reaction stoichiometry.20 The reaction solution of arbutin with ABTS radical cation changed to a purple color, whereas the reaction solution of hydroquinone with ABTS radical cation changed from dark green to light green. Generally, the characteristic color of ABTS radical cation is discolored from dark green to light green when the radical is bleached by antioxidants. Therefore, the formation of a purplecolored compound seems to be related to the high stoichiometry of arbutin in the ABTS radical cation-scavenging assay. To investigate characteristics of the radical-scavenging reaction previously reported,20 the reaction mixture of arbutin with ABTS radical cation was analyzed by HPLC. The HPLC profile of the reaction mixture of arbutin (20 μM) and ABTS radical cation (100 μM) for 120 min in 50 mM citrate buffer (pH 6) is shown in Figure 2A. The peak of arbutin at tR 7 min had disappeared, whereas the peaks of major reaction products were found at tR 28 and 32 min. To effectively obtain major reaction products, the reactions of arbutin at concentrations in the range of 167−500 μM and ABTS radical cation at 1.0 mM were investigated in 50 mM citrate buffer (pH 6) at room temperature for 15 min (Figure 3). Figure 2B shows a representative HPLC chromatogram of the reaction mixtures. Under the conditions used, the same products as the reaction products under the ABTS radical cation-scavenging assay conditions were obtained (Figure 2A). Ultraviolet and visible spectra of the two reaction products were obtained using the diode array detector attached to the HPLC system (data not shown). As judged from the spectra, peak 1 at tR 28 min was a purple-colored compound. The formation of a purple-colored compound increased with an increase in the concentration of arbutin from 167 to 250 μM and decreased at concentrations of >333 μM (Figure 3). On the other hand, small residual amounts of arbutin were observed when 167, 200, and 250 μM of arbutin were used in the reaction, and the residual amount sharply increased at concentrations of >333 μM. These results suggested that the optimal reaction conditions for obtaining a

purple-colored compound were 250 μM arbutin and 1.0 mM ABTS radical cation. We tried to isolate a purple-colored compound (peak 1; tR 28 min, Figure 2B) and the other compound (peak 2; tR 32 min, Figure 2B) from the reaction mixture of arbutin (102.1 mg, 0.375 mmol) and ABTS radical cation (1.5 mmol) in 1500 mL of 50 mM citrate buffer (pH 6). The reaction mixture was concentrated to about 350 mL and was then chromatographed on a Diaion HP20 column and on a Toyopearl HW-40C column to give a purple powder, 3 (146.2 mg). The other product was further chromatographed on a Toyopearl HW-40F column and on a Wakogel 50NH2 column to give a white powder, 4 (48.7 mg). The reaction products, 3 and 4, were fully characterized and identified by the mass spectrum and several NMR spectra (1H, 13C, 1H−1H COSY, HSQC, and HMBC). The 1H NMR spectrum of reaction product 3 showed ABX type signals (δ 6.84, 7.23, and 7.42) and ethyl proton signals (δ 0.94 and 3.74) in the ABTS unit and signals due to three olefin protons (δ 5.24, 6.02, and 6.09) and glucose protons in the arbutin unit. 13C resonances of the arbutin unit displayed a ketone carbon at δ 183.5, three olefin carbons at δ 99.0, 129.3, and 138.1, and two quaternary sp2 carbons bearing a heteroatom at δ 147.2 and 153.1, besides six glucose carbons. The HMBC spectrum of reaction product 3 showed correlations of H-3′ (δ 6.09) with C-1′ (δ 183.5), C-4′ (δ 153.1), and C-5′ (δ 138.1). The H-5′ signal (δ 6.02) exhibited correlations with signals of C-1′, C-3′ (δ 99.0), and C-4′, and the signal of H-6′ (δ 5.24) was correlated with C-2′ (δ 147.2) and C-4′. The position of the glycosidic linkage was determined to be C-4′ as manifested by the HMBC correlations observed between the anomeric proton H-1″ (δ 4.41) and C-4′. Furthermore, these NMR data and high-resolution ESI-MS data indicated that the quaternary sp2 carbon assigned to C-2′ was attached to the nitrogen atom in the ABTS degradation fragment through a double bond. On the basis of these findings, the structure of 3 was determined as shown in Figure 4. The reaction product 4 was identified by comparing the spectral data with reported data.27 Therefore, 3 and 4 were determined to be a covalent adduct of arbutin with an ABTS degradation fragment and 3-ethyl-6-sulfonate benzothiazolone, respectively (Figure 4). Osman et al.27 reported that two degradation products, 3-ethyl-6-sulfonate benzothiazolone and 3-ethyl-6sulfonate benzothiazolinone imine, were obtained from incubation of the ABTS radical cation with the polyphenols 7287

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of the adduct suggests that the adduct is most likely formed by reaction via intermediate formation of arbutin with ABTS radical cation. At 15 min, the amounts of an arbutin−ABTS degradation fragment adduct and 3-ethyl-6-sulfonate benzothiazolone that were formed were both ca. 190 μM, and the amount of arbutin consumed was 200 μM. Thus, 95% of arbutin contributed to the adduct formation. These results indicated that the formation of an arbutin−ABTS degradation fragment adduct and 3-ethyl-6-sulfonate benzothiazolone was the main reaction pathway for scavenging ABTS radical cation. A time-course study of the radical-scavenging reactions of arbutin (20 μM), an arbutin−ABTS degradation fragment adduct (60 μM), and 3-ethyl-6-sulfonate benzothiazolone (60 μM) against ABTS radical cation (100 μM) was carried out under an ABTS radical cation-scavenging assay condition (Figure 6). The reaction of arbutin rapidly occurred within 5

Figure 4. Proposed structures of two reaction products.

(+)-catechin, (−)-epicatechin, and phloroglucinol in acetate buffer (pH 5). The formation of 3-ethyl-6-sulfonate benzothiazolone in the reaction of arbutin with ABTS radical cation was the same as that in the case of polyphenols, but the adduct formation of an antioxidant with an ABTS degradation fragment was not the same. We also observed that α-arbutin,28 a synthetic arbutin anomer, scavenged the ABTS radical cation to give two reaction products. The reaction products seemed to be an isomer of 3 and 4 by HPLC−diode array detection (data not shown). These results suggested that arbutin scavenged the ABTS radical cation to form the arbutin radical and that the temporary formation of the arbutin radical may be associated with the production of an arbutin−ABTS degradation fragment adduct. To investigate whether the formation of an arbutin−ABTS degradation fragment adduct and 3-ethyl-6-sulfonate benzothiazolone was the main reaction pathway of arbutin for scavenging ABTS radical cation, we carried out time-course studies on the formation amounts of an arbutin−ABTS degradation fragment adduct and 3-ethyl-6-sulfonate benzothiazolone and on the consumption amounts of arbutin in the case of the reaction between arbutin (200 μM) and ABTS radical cation (1.0 mM) (Figure 5). In the first 5 min of the

Figure 6. Time course of ABTS radical cation-scavenging reactions of arbutin−ABTS degradation fragment adduct, 3-ethyl-6-sulfonate benzothiazolone, and arbutin: (△) arbutin−ABTS degradation fragment adduct (60 μM); (●) 3-ethyl-6-sulfonate benzothiazolone (60 μM); (◇) arbutin (20 μM); (◆) control. Each value is the mean ± SD of three separate experiments.

min and slightly proceeded in the period of 5−120 min. The reaction of an arbutin−ABTS degradation fragment adduct proceeded only very slightly, whereas the reaction of 3-ethyl-6sulfonate benzothiazolone proceeded slightly and continuously for 120 min. The stoichiometric factor RSA(n) was defined as the number of radicals consumed per molecule of each antioxidant at 120 min. The RSA(n) values of arbutin, an arbutin−ABTS degradation fragment adduct, and 3-ethyl-6sulfonate benzothiazolone were 3.2, 0.0, and 0.2, respectively. An arbutin−ABTS degradation fragment adduct is considered to be the final product in the radical-scavenging reaction because the arbutin−ABTS degradation fragment adduct scavenged very little ABTS radical cation. The RSA(n) value of arbutin at 5 min was 3.0, and arbutin was almost completely consumed within the first 5 min of the radical-scavenging reaction (Figure 5), although the RSA(n) value of arbutin at 120 min was 3.2, suggesting that one molecule of arbutin scavenges three ABTS radical cation molecules. The increment in the RSA(n) value of arbutin from 5 to 120 min is consistent with the RSA(n) value of 3-ethyl-6-sulfonate benzothiazolone for 120 min. Thus, the increase in the RSA(n) value of arbutin from 5 to 120 min was caused by a side reaction of 3-ethyl-6sulfonate benzothiazolone generated in the radical-scavenging reaction of arbutin. These results suggested that one molecule of arbutin scavenges three ABTS radical cation molecules to generate an arbutin−ABTS degradation fragment adduct as a final reaction product.

Figure 5. Time course of the formation of an arbutin−ABTS degradation fragment adduct and 3-ethyl-6-sulfonate benzothiazolone and the consumption of arbutin. Arbutin (200 μM) reacted with ABTS radical cation (1.0 mM) in sodium citrate buffer (50 mM, pH 6): (△) Arbutin−ABTS degradation fragment adduct; (●) 3-ethyl-6-sulfonate benzothiazolone; (◇) arbutin. Values are means ± SD of three separate experiments.

reaction, an arbutin−ABTS degradation fragment adduct and 3ethyl-6-sulfonate benzothiazolone were rapidly formed, and the amounts formed were constant in the period of 5−15 min. The formation profile of an arbutin−ABTS degradation fragment adduct agreed closely with that of 3-ethyl-6-sulfonate benzothiazolone. Arbutin was almost completely consumed within the first 5 min of the reaction. The consumption rate was slightly faster than the formation rate of an arbutin−ABTS degradation fragment adduct and 3-ethyl-6-sulfonate benzothiazolone. The consumption of arbutin prior to the formation 7288

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Figure 7. Scheme of ABTS radical cation-scavenging reaction of arbutin.

Hydroquinone is a potent antioxidant having two oxidizable hydroxyl groups on the benzene ring. In our previous study, it was found that one molecule of hydroquinone scavenged two ABTS radical cation molecules.20 Because arbutin, a glucoside of hydroquinone, has only one oxidizable hydroxyl group of hydroquinone, the antioxidant activity was thought to be decreased compared with that of hydroquinone. However, arbutin exhibited strong antioxidant activity superior to that of hydroquinone in an ABTS radical cation-scavenging assay. Thus, the reaction stoichiometry of arbutin was much higher than that of hydroquinone. In this study, we investigated the reaction mechanism of arbutin against ABTS radical cation that caused high stoichiometry of arbutin in an ABTS radical cationscavenging assay. HPLC analysis of the reaction mixture of arbutin and ABTS radical cation indicated the existence of two reaction products. The two reaction products were purified and identified to be a covalent adduct of arbutin with an ABTS degradation fragment and 3-ethyl-6-sulfonate benzothiazolone. Time-course studies for the quantitative formation of an arbutin−ABTS degradation fragment adduct and 3-ethyl-6sulfonate benzothiazolone and for the quantitative consumption of arbutin showed that the formation of an arbutin−ABTS degradation fragment adduct and 3-ethyl-6-sulfonate benzothiazolone was the main reaction pathway of arbutin for scavenging the ABTS radical cation. A time-course study of the radical-scavenging reactions of arbutin and the two reaction products suggested that one molecule of arbutin scavenges three ABTS radical cation molecules to generate an arbutin− ABTS degradation fragment adduct as a final reaction product. Our results suggest that one molecule of arbutin reduced two ABTS radical cation molecules to ABTS and then cleaved the third ABTS radical cation molecule to generate two products, an arbutin−ABTS degradation fragment adduct and 3-ethyl-6sulfonate benzothiazolone, although the detailed reaction mechanism for the adduct formation remains unknown (Figure 7). Recently, it has been reported that arbutin protected cells from X-irradiation-induced apoptosis by reducing intracellular hydroxyl radical production29 and that arbutin had a protective effect against cyclosporine A-induced toxicity by preventing an increase in serum lipid peroxidation.30 Therefore, arbutin is expected to act as an antioxidant for health and skin care purposes as well as a skin-lightening agent for cosmetic purposes.

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AUTHOR INFORMATION

Corresponding Author

*(A.T.) Fax: +81-824-74-1779. E-mail: [email protected]. jp. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to the SC-NMR Laboratory of Okayama University and the MS Laboratory of the Faculty of Agriculture at Okayama University.

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REFERENCES

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DOI: 10.1021/acs.jafc.6b02847 J. Agric. Food Chem. 2016, 64, 7285−7290

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DOI: 10.1021/acs.jafc.6b02847 J. Agric. Food Chem. 2016, 64, 7285−7290