Metmyoglobin Reduction by Polyphenols and Mechanism of the

Article pubs.acs.org/JAFC

Metmyoglobin Reduction by Polyphenols and Mechanism of the Conversion of Metmyoglobin to Oxymyoglobin by Quercetin Miyuki Inai,† Yukari Miura,† Sari Honda,† Akiko Masuda,‡ and Toshiya Masuda*,† †

Graduate School of Integrated Arts and Sciences, University of Tokushima, Tokushima 770-8502, Japan Faculty of Human Life Science, Shikoku University, Tokushima 771-1192, Japan

‡

S Supporting Information *

ABSTRACT: The effect of antioxidant polyphenols and related phenolic compounds from plants on the reduction of metmyoglobin (MetMb) was investigated. Potent activity in the reduction of MetMb to oxymyoglobin (MbO2), a bright red protein in meat, was observed for three flavonols, kaempferol, myricetin, and quercetin, at 300 μmol/L against 60 μmol/L MetMb. Sinapic acid, catechin, nordihydroguaiaretic acid, taxifolin, morin, and ferulic acid promoted reduction at 600 μmol/L. A mechanism for the reduction by one of the active flavonols, quercetin, was proposed on the basis of analytical results for redox reaction products derived from quercetin. This suggested the importance of a high propensity toward reduction of the flavonol structure and rapid convertibility of the quinone form to the phenol form for the MbO2 reduction and the maintenance of the level of MbO2 produced. KEYWORDS: metmyoglobin, oxymyoglobin, polyphenol, reducing activity, quercetin, flavonol

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INTRODUCTION

polyphenol, it is a potent bioreductant and can reduce MetMb to afford MbO2 in air.10,11 Ashida et al.12 have reported inhibition of MbO2 oxidation by relatively low gallic acid concetrations. Mordent et al.13 have reported MetMb reduction by high ubiquinol-1 concentrations. Moreover, various thiols were examined for MetMb reduction, and only dihydrolipotic acid was found to be active.14 Khalife and Lupidi15 have found that thymoquinone, an oxidized form of thymol, reduces MetMb in the presence of glutathione (GSH) or reduced nicotinamide adenine dinucleotide (NADH). Carnosine, a peptide in muscles, has been reported to reduce MetMb to MbO2.16 In this investigation, we studied the effect of 20 kinds of polyphenols and related phenolic compounds found in edible plants as antioxidants on MetMb reduction and also clarified the mechanism of MetMb reduction by a potently active polyphenol, quercetin.

The red color of fresh meat is because of the muscle heme protein, myoglobin (Mb), and the color of Mb varies with the redox state of heme. The bright red color of meat, which is desirable to consumers, is due to oxymyoglobin (MbO2) formed in meat blooming. It is well-known that MbO2 is relatively unstable, because it readily oxidizes to metmyoglobin (MetMb). MetMb has a brownish color that reduces the market value of meat. Controlling MbO2 oxidation is very important to maintain the color of fresh meat and has been a persisting problem in food science.1 Many physical and chemical techniques have been developed to prevent browning of meat involving the use and manipulation of various gases.2−4 These techniques, however, have problems because of food safety and expense. Reduction of brown MetMb to red MbO2 should be considered as an alternative technique to maintain the bright red color of fresh meat. Bekhit and Faustman5 have summarized many enzymatic and non-enzymatic MetMb reduction methods. The brown color of the meat is not apparent if 60% MetMb in the meat is reduced.6 A recent survey also indicated that consumers recognize the red color of meat at an MbO2/MetMb ratio over 3.5.7 Therefore, MetMb reduction should contribute to the preservation of the color of meat.8 Polyphenols are well-known bioactive constituents of plants and plant-derived foods. They exert potent antioxidant activity against oxidative deterioration of food constituents by various mechanisms, including radical trapping, removal of transition metals, and reduction ability. We have been interested in the antioxidant activity of polyphenols and their potency as reductants for the control of the redox state of Mb. In a previously reported study,9 we used polyphenols and related phenolic compounds to prevent MbO2 oxidation in vitro; however, some potent antioxidant polyphenols actually promoted oxidation. Although ascorbic acid is not a © 2014 American Chemical Society

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MATERIALS AND METHODS

Chemicals and Instruments. Myoglobin (from the horse heart), quercetin (purity > 95%, as dihydrate), gentisic acid (purity > 98%), protocatechuic acid (purity > 97%), L-ascorbic acid (purity > 99.5%), potassium hexacyanoferrate (III), and sodium hydrosulfite were obtained from Nacalai Tesque (Kyoto, Japan). Caffeic acid (purity > 98%), ferulic acid (purity > 98%), morin (purity > 98% as hydrate), sinapic acid (purity > 99%), luteolin (purity > 98%), hydroxytyrosol (purity > 98%), resveratrol (purity > 98%), and myricetin (purity > 97%) were purchased from Tokyo Kasei (Tokyo, Japan). (+)-Catechin (purity > 98%, as hydrate), rosmarinic acid (purity > 97%), chlorogenic acid (purity > 95%), nordihydroguaiaretic acid (purity > 97%), syringic acid (purity > 95%), dihydrocaffeic acid (purity > 98%), and Chelex100 resin were obtained from Sigma-Aldrich (St. Louis, Received: Revised: Accepted: Published: 893

September 30, 2013 January 7, 2014 January 8, 2014 January 8, 2014 dx.doi.org/10.1021/jf404357h | J. Agric. Food Chem. 2014, 62, 893−901

Journal of Agricultural and Food Chemistry

Article

Figure 1. Chemical structures of the polyphenols and related phenols investigated for MetMb reduction. Preparation of MbO2 and MetMb. MbO2 was prepared using the method reported previously.9 MetMb solution was prepared from a stock solution of MbO2. Potassium hexacyanoferrate (III) (2 mg) was added to a solution of MbO2 (120 μmol/L) in a phosphate buffer (pH 7.4, 3 mL) at 23 °C. After gentle stirring for 0.5 min, the solution was desalted 3 times at 14000g for 6 min at 4 °C with an Amicon Ultra-0.5, Ultracel-10 ultrafiltration membrane (Merck Millipore, Cork, Ireland) using phosphate buffer. The final residual solution (ca. 0.5 mL) was diluted with buffer, yielding 120 μmol/L MetMb solution (3 mL). The purity and concentration of the MetMb solution were confirmed with a UVmini 1240 ultraviolet−visible (UV−vis) spectrometer (Shimadzu).17 Measurement of MetMb Reduction and MbO2 Oxidation. To a 96-well microplate, 50 mmol/L phosphate buffer (pH 7.4, 140 μL), 120 μmol/L MetMb or MbO2 in the same buffer (150 μL), and an appropriate amount of test samples (polyphenols, their products, or L-

MO). Vanillic acid (purity > 96%) was purchased from Wako Pure Chemicals (Osaka, Japan). Kaempferol (purity > 97%) and taxifolin (purity > 96%) were obtained from Funakoshi (Tokyo, Japan). All solvents [extra pure grade or high-performance liquid chromatography (HPLC) grade] were obtained from Nacalai Tesque. Nuclear magnetic resonance (NMR) spectra were obtained from an ECS-400 spectrometer (JEOL, Tokyo, Japan) using the pulse sequences of the manufacturer. High-resolution mass spectrometric data were obtained using a XEVO QtofMS spectrometer (Waters Japan, Tokyo, Japan) in negative electrospray ionization (ESI) mode. A PU-2089 low-pressure gradient system (JASCO, Tokyo, Japan) equipped with a MD-2018 photodiode array (PDA) detector was employed for analytical HPLC. PDA data were analyzed using ChromNAV, version 1.8 (JASCO). A LC-6AD system (Shimadzu, Kyoto, Japan) equipped with a UV-970 detector (JASCO) was used for preparative HPLC. 894

dx.doi.org/10.1021/jf404357h | J. Agric. Food Chem. 2014, 62, 893−901


Article

Figure 2. Effects of phenols (600 or 300 μmol L−1) on MetMb reduction. Results are expressed as the mean ± standard deviation (SD) (n = 6). (a) Activity of kaempferol, luteolin, myricetin, and quercetin was measured at 300 μmo/L because their solubilities are below 600 μmol/L. (b) Data on myricetin after 1 h are low of accuracy because of the dark brown color of the solution mixture. ascorbic acid) in dimethyl sulfoxide (DMSO) (10 μL) were subsequently added. The microplate was incubated at 37 °C in a Multiskan GO microplate reader (Thermo Fisher Scientific, Yokohama, Japan) for 3 h, and absorbance at 500 and 582 nm was measured at 1 h intervals. At the beginning and end of the experiment, visible spectra (450−650 nm) were obtained with the same microplate reader. MbO2 and MetMb concentrations in the well were calculated by the following equation based on the absorption spectra of 60 μmol/ L pure MbO2 or MetMb: MbO2 (μmol/L) = (89.7A582) − (32.9A500), where A500 and A582 refer to absorbance at 500 and 582 nm, respectively. Redox conversion efficiency between MetMb and MbO2 was expressed as a percentage calculated by the MbO2 concentration relative to the initial concentrations (60 μmol/L) of each MbO2 and MetMb. HPLC and Liquid Chromatography−Mass Spectrometry (LC−MS) Analyses of Quercetin Products during MetMb Reduction. At 1 h intervals, an aliquot (5 μL) was removed from the above-mentioned reaction with quercetin (300 μmol/L) and the

reaction products were analyzed under the following conditions: column, a 250 × 4.6 mm inner diameter, 5 μm, Cosmosil 5C18-AR-II (Nacalai Tesque); flow rate, 1.0 mL/min; solvent A, 1% acetic acid in water; solvent B, acetonitrile; gradient conditions, linear gradient from 5 to 100% solvent B (40 min); and detection, absorbance at 280 nm. The concentration of each product in the reaction mixture was calculated using the following calibration equations: quercetin, Y (μmol/L) = 0.70X × 10−3 + 0.15 (range for Y, 50−300 μmol/L); product I, Y (μmol/L) = 3.62X × 10−4 + 2.39 (range for Y, 1−1000 μmol/L); product II, Y (μmol/L) = 1.02X × 10−3 + 1.64 (range for Y, 10−1000 μmol/L); product III, Y (μmol/L) = 3.59X × 10−4 + 8.94 (range for Y, 10−1000 μmol/L), where X represents the peak area at 280 nm. The MS data for each product was obtained by LC−MS. An additional aliquot (5 μL) was also taken after 1 and 3 h for the analysis of products I−III, and the analyses were carried out under the following conditions: separation conditions, column, a 250 × 4.6 mm inner diameter, 5 μm, Cosmosil 5C18-AR-II (Nacalai Tesque); flow rate, 0.5 mL/min; solvent A, 1% acetic acid in ultrapure water, solvent 895



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Figure 3. HPLC profile for the reaction of quercetin and MetMb at 2 h. °C, and absorption at 582 and 500 nm was obtained at 1 h intervals. At the same intervals, aliquots (5 μL) were taken and injected into the HPLC unit. Analysis of the reaction products I−III was carried out under the following conditions: column, a 250 × 4.6 mm inner diameter, 5 mm, Cosmosil 5C18-AR-II (Nacalai Tesque); flow rate, 1.0 mL/min; solvent A, 1% acetic acid in water; solvent B, acetonitrile; linear gradient from 5 to 100% solvent B (40 min); and detection, absorbance at 280 nm. The concentration of each product in the reaction mixture was calculated using the above-mentioned equations.

B, acetonitrile (LC−MS grade, Merck, Darmstadt, Germany); gradient conditions, linear gradient from 5 to 100% solvent B (80 min); and MS conditions, mode, negative ESI; capillary voltage, 2.4 kV; cone voltage, 40 V; source temperature, 150 °C; desolvation temperature, 500 °C; cone gas flow rate, 50 L/h; desolvation gas flow rate, 1000 L/ h, MSE low collision energy, 6 V; and MSE high collision energy, 20− 30 V. The elemental composition of each peak compound was calculated from the high-resolution MS data of the deprotonated molecular ion using MassLynx software, version 4.1 (Waters). Preparation and Structure Elucidation of Quercetin Oxidation Products I−III. To a solution of quercetin (50 mg) in acetonitrile (50 mL) and water (50 mL) was added 90 mg of FeCl3· 6H2O and the solution was stirred at 37 °C for 1 h. The solution was passed through a short column of Chelex100 (10 g), and the eluate was evaporated in vacuo. The residue obtained was purified by preparative HPLC under the following conditions: column, a 250 × 20 mm inner diameter, 5 μm, Cosmosil 5C18-AR-II (Nacalai Tesque); flow rate, 9.0 mL/min; solvent, 0.1% trifluoroacetic acid in 85:15 (v/v) water/acetonitrile. All eluates from 18−24 min were collected and evaporated to give product I (21, 40 mg). Product I (21): HR-ESIMS (m/z) [M − H]−, calcd for C15H9O8, 317.0297; found, 317.0284. UV λmax (acetonitrile/H2O/acetic acid), 325 (sh), 292 nm. 1H NMR (acetone-d6) δ: 7.64 (1H, d, J = 2.4 Hz), 7.57 (1H, dd, J = 8.1 and 1.8 Hz), 6.92 (1H, d, J = 8.1 Hz), 6.13 (1H, brd, J = 1.2 Hz), 6.12 (1H, brd, J = 1.2 Hz) [6.13 (2H, brs)]. Products II (22, 1 mg) and III (23, 7 mg) were also isolated after oxidizing quercetin (55 mg) for 2 h with FeCl3·6H2O (99 mg) in acetonitrile−water [1:1 (v/v), 100 mL] at room temperature. Purification was carried out as follows: The reaction solution was passed through a Chelex100 column (10 g), and the eluate was purified by preparative HPLC after evaporation. Two peaks (14−16 and 22−25 min) were collected and evaporated to give products II (22) and III (23), respectively. Product II (22) (protocatechuic acid): ESIMS (m/z) 153 [M − H]− (100%). UV λmax (acetonitrile/H2O/acetic acid), 292, 260 nm. 1H NMR (DMSOd6) δ: 8.30 (1H, br s), 7.31 (1H, d, J = 1.8 Hz), 7.26 (1H, J = 8.2 and 1.8 Hz), 6.76 (1H, J = 8.2 Hz). Product III (23) (2,4,6trihydroxyphenylglyoxylic acid): ESIMS (m/z), 193 [M − H]− (40%), 195 (40%), 177 (50%), 153 (100%). UV λmax (acetonitrile/ H2O/acetic acid), 292 nm. 1H NMR (DMSO-d6) δ: 5.62 (2H, s). HPLC Analysis of the Reactions of Products I−III with MetMb. To each well of a microplate, 140 μL of 50 mmol/L phosphate buffer (pH 7.4), 10 μL of the appropriate concentration of each product in DMSO, and 150 μL of 120 μmol/L of MetMb or MbO2 in the same buffer were added. The plate was incubated at 37

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RESULTS AND DISCUSSION MetMb Reduction by Polyphenols. Ascorbic acid is known to potently reduce MetMb; however, the precise

Figure 4. Time-course analytical data for products I−III in the reaction of quercetin and MetMb.

concentration required for this reduction in vitro has not yet been reported. First, we examined the effect of the ascorbic acid concentration (60−2400 μmol/L) on MetMb (60 μmol/L) in pH 7.4 phosphate buffer. The results obtained showed that 600 μmol/L ascorbic acid reduced MetMb gradually, forming MbO2 equivalent to 80% of the initial amount of MetMb after 3 h at 37 °C. Therefore, these conditions were employed in experiments using 20 kinds of polyphenols and related phenolic compounds (Figure 1). Figure 2 shows the MetMb reduction 896



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and MbO2 formation by the polyphenols. The data are shown in percentage of MbO2 in total myoglobins, which was calculated on the basis of absorbance at 500 and 582 nm of each 60 μmol/L pure MbO2 and MetMb.18 Although most polyphenols showed weak reducing activity as reductants, sinapic acid (17) (600 μmol/L) exhibited apparent potency, followed by catechin (2), nordihydroguaiaretic acid (12), taxifolin (19), morin (10), and ferulic acid (5). Unfortunately, the activity of some flavonoid polyphenols could not be measured at this concentration because of insolubility. Therefore, the activity of 300 μmol/L of these flavonoids [kaempferol (8), luteolin (9), myricetin (11), and quercetin (14)] was measured; the results are illustrated in the lower part of Figure 2. As shown there, kaempferol (8), quercetin (14),

Figure 5. Chemical structures of products I−III.

Figure 6. MetMb reduction andMbO2 oxidation of quercetin and product I and changes in concentrations of quercetin and product I in the reaction solutions. Panels A and E refer to MetMb reduction. Panels B and F refer to MbO2 oxidation. Panels C and G express concentration changes during MetMb reduction. Panels D and H express concentration changes during MbO2 oxidation to MetMb. Data are expressed as the mean ± SD; note that most SD values are smaller than the symbols used in the graph. 897



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Figure 7. MetMb reduction and MbO2 oxidation of products II and III and changes in concentrations of products II and III in the reaction solutions. Panels A and E refer to MetMb reduction. Panels B and F refer to MbO2 oxidation. Panels C and G express concentration changes during MetMb reduction. Panels D and H express concentration changes during MbO2 oxidation to MetMb. Data are expressed as the mean ± SD; note that most SD values are smaller than the symbols used in the graph.

(products I−III) coinciding with the decrease in quercetin levels during the reaction. The formation of product I was faster than that of products II and III, indicating that product I was the first oxidation product from quercetin and that products II and III were secondary products, possibly derived from product I. The MetMb reduction was completed by 1 h, and the produced MbO2 remained at the same concentration (the bottom−right panel of Figure 2). Product I reached a plateau after 2 h, while products II and III continued to increase and quercetin decreased over 3 h. These results suggested that quercetin was largely responsible for MetMb reduction, while the other products probably contributed to the maintenance of MbO2 levels. Identification of Products I−III from Quercetin. To identify the structures of products I−III, we applied various

and myricetin (11) showed apprant reducing activity even at this lower concentration. These compounds are flavonols, and flavonols are known to be potent antioxidants19 with low reduction potential20 relative to other flavonoids. Next, we undertook a chemical investigation of the high reducing activity of the flavonol, quercetin (14). HPLC Analysis of Quercetin Products during MetMb Reduction. On the basis of redox principles, quercetin should be oxidized during MetMb reduction. We carried out a timecourse analysis of the MetMb reducing reaction of quercetin by HPLC. Figure 3 shows analytical results at 2 h. In the HPLC profile, three noticeable peaks were observed at retention times of 7.8, 8.3, and 11.3 min, in addition to quercetin (17.8 min). The time course of the formation of these reaction products is summarized in Figure 4. Figure 4 shows three new compounds 898



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Figure 8. Proposed mechanism for MbO2 production from MetMb by quercetin.

other. Also, the chemical shifts of the 1H NMR signals for 6 and 8 positions were observed at slightly different values depending up the on concentration. Tautomerism in product I has been reported by several groups,22,23,25−27 but the observed UV spectrum of product I suggested that it exists mainly as structure I (21) (Figure 5). Effect of Quercetin and Its Oxidation Products on the Redox State of Mb. The effects of three isolated quercetin oxidation products on the redox state of Mb were examined and compared to quercetin. Results for the change in the concentration of MbO2 in the presence of 60−300 μmol/L quercetin are shown in Figure 6. Panels A and B of Figure 6 show that quercetin oxidizes MbO2 and reduces the MetMb concentration independently; both reactions converged to ca. 40% MbO2. Although quercetin and Mb seemed to be under redox equilibrium, quantitative analysis of the reaction mixture (panels C and D of Figure 6) showed that quercetin decreased continuously after reaching equilibrium. These results indicate that the oxidation products identified also influence the redox

chemical reagents to quercetin. We found that oxidation by ferric chloride afforded better results than other oxidation reagents. Treatment of quercetin with ferric chloride in acetonitrile and water gave a mixture of the same products as observed in the MetMb reduction. The products were purified by preparative HPLC using an ODS column to give products I−III. 1H NMR, UV, and MS analysis of these products revealed that they are products of oxidation and oxidative degradation of quercetin. The structures of these products are shown in Figure 5. Products II (22) (protocatechuic acid) and III (23) (2,4,6-trihydroxyphenylglyoxylic acid) were previously identified from peroxidase oxidation of quercetin, and their UV spectra were identical with the reported values.21 Although product I was also identified previously from the oxidation of quercetin22−24 and the structure is tentatively identified as product I (21) in Figure 5, it may exist as a tautomer because HPLC of product I showed two slightly split peaks; however, the corresponding pure forms could not be isolated under the analytical conditions because of their rapid conversion to each 899



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polyphenols to maintain the fresh meat color as reductants and also to develop a new color-maintaining technique for meat foods based on the redox chemistry of polyphenols.

state of Mb. Panels E−H of Figure 6 and panels A−H of Figure 7 show the redox activity of these products toward MbO2 and MetMb, and their concentration changes during the reaction. As shown in panels E and F of Figure 6, product I also oxidizes MbO2 and reduces MetMb, but efficiency is much lower than quercetin in both cases. Products II and III have negligible oxidative activity toward MbO2 and no reductant activity toward MetMb (panels A, B, E, and F of Figure 7). These results for the redox relation between myoglobins and these products were confirmed by HPLC analysis of products I−III in the reaction mixture (panels G and H of Figure 6 and panels C, D, G, and H of Figure 7). As shown in panels G and H of Figure 6, the concentration of product I (300 μmol/L) was decreased with myoglobins, revealing that product I influences the redox reactions with myoglobins. On the other hand, treatment of products II and III with MetMb and MbO2 shows a negligible change in the concentration, indicating that products II and III are stable under these reaction conditions and do not strongly influence the redox state of Mb. Proposed Mechanism for MetMb Reduction by Quercetin. We proposed a mechanism for MetMb reduction by quercetin in vitro (Figure 8). First, quercetin reduces MetMb to produce bright red MbO2; however, oxidized quercetin drivatives, including the B-ring quinone (24), enhance MbO2 oxidation. It has been found that electrophilic compounds, such as α,β-unsaturated aldehydes enhanced MbO2 oxidation.28 These electrophiles were indicated to react with Mb at nucleophilic amino acid residues, destabilizing MbO2.29 Quinone derivatives of polyphenols also have the same electrophilic tendency30,31 to alter the tertiary structure of proteins, such as MbO2, which is rich in nucleophilic amino acid residues, such as histidine and lysine.32 Polyphenols are potent antioxidants bearing catechol moieties, which during oxidation afford ortho-quinone derivatives, and some polyphenols afford quinone derivatives that accumulate in the system.33,34 To date, no quinone derivative has been isolated from quercetin or related flavonols, but several water adducts have been identified.24,35,36 These results suggested that water addition to the oxidized flavonols is fast and quickly yields phenolic compounds that prevent quinone−protein interactions. As illustrated in Figure 8, one molecule of water is added to a tautomer of the quinone (25) and affords product I (21) via water adducts 26 and 27, which has a catechol moiety that can reduce MetMb. Although reduction by product I (21) also produces another quinone derivative 28 that may oxidize MbO2, its efficiency is much lower than that of quercetin. In addition to its low redox activity, product I (21) is converted to products II (22) and III (23) via oxidation and subsequent hydrolysis. Thus, products II (22) and III (23) exhibit almost no redox activity toward MetMb or MbO2. These properties are also supported by the higher reduction potential of product II (E7 of 0.60 V)37 and that of quercetin (E7 of 0.33 V).38 From the results of this investigation, we propose that most of the predominant MetMb-reducing activity of quercetin and related flavonols is because of their high propensity to reduce MetMb combined with the rapid conversion of their oxidized products, which destabilize MbO2, to redox-stable compounds. The results of this investigation showed that some polyphenols were applicable to reduce MetMb to bright red MbO2. The bright red color is very important to maintain the freshness and quality of meat and meat-derived foods. The proposed reduction mechanism of one of polyphenols, quercetin, have revealed high potential for application of

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ASSOCIATED CONTENT

S Supporting Information *

Figure for the concentration effect on MetMb reduction by Lascorbic acid. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Telephone/Fax: +81-88-656-7244. E-mail: masuda.ias@ tokushima-u.ac.jp. Funding

Financial support was from the Japan Society for the Promotion of Science (KAKENHI 23500939). Notes

The authors declare no competing financial interest.

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REFERENCES

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Metmyoglobin Reduction by Polyphenols and Mechanism of the

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