Reducing Effects of Polyphenols on Metmyoglobin and the in Vitro

Article pubs.acs.org/JAFC

Reducing Effects of Polyphenols on Metmyoglobin and the in Vitro Regeneration of Bright Meat Color by Polyphenols in the Presence of Cysteine Yukari Miura,† Miyuki Inai,† 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

‡

ABSTRACT: The effect of polyphenols and related phenolic compounds on the reduction of metmyoglobin (MetMb) to oxymyoglobin (MbO2), in the presence of cysteine, was investigated. Caffeic acid, dihydrocaffeic acid, and hydroxtyrosol (600 μmol/L) did not show any reducing activity individually. However, their highly potent activity in the reduction of MetMb to MbO2 was observed in the presence of equimolar amounts of cysteine. On the basis of the analytical results for the redox reaction products generated during the MetMb-reducing reaction of caffeic acid, we proposed a mechanism for the polyphenol-mediated reduction of MetMb. As per the proposed mechanism, the antioxidant polyphenols having a catechol substructure can effectively reduce MetMb to MbO2 with chemical assistance from nucleophilic reactive thiol compounds such as cysteine. Moreover, cysteine-coupled polyphenols such as cysteinylcaffeic acids (which are coupling products of caffeic acid and cysteine) can be used as preserving agents for retaining the fresh meat color, because of their powerful reducing effect on MetMb. The reduction of MetMb to MbO2 changes the color of meat from brown to the more desirable bright red. KEYWORDS: metmyoglobin, oxymyoglobin, cysteinylcaffeic acid, polyphenol, meat color preservation

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that two flavonols (kaempferol and quercetin) could partially convert MetMb into MbO2. However, other polyphenols, regardless of their reducing potency, could not produce sufficient amounts of MbO2. This insufficiency may be attributed to their low reducing activity or to the pro-oxidant property of polyphenols. Khalife and Lupidi9 have reported that a quinone derivative from thymol reduces MetMb in the presence of glutathione. The addition of equimolar amounts of cysteine to a polyphenol was found to slow down the rate of MbO2 oxidation.3 Several cysteine-coupled polyphenols were also found to enhance antioxidant capacity against lipid oxidation.10,11 These results indicate that combining a cysteine or a related thiol compound with polyphenols influences the redox state of myoglobin, thereby offering a viable alternative for coloration and retaining the bright red color of meat. In this investigation, the effect of polyphenols on the reduction of MetMb to MbO2 in the presence of cysteine was studied.

INTRODUCTION The bright red color of meat is desirable to most consumers as an indicator of the freshness of meat. The bright red color of meat is the result of oxymyoglobin (MbO2), an oxygencoordinated myoglobin containing ferrous ion. Myoglobin (Mb) is a muscle heme protein that exhibits various redox states, depending on the state of the chelated Fe ion. Mb is rapidly converted to MbO2 in the presence of molecular oxygen. This phenomenon is important for the meat industry because meat gains its bright red color from MbO2. However, the produced MbO2 is not very stable and is readily oxidized to metmyoglobin (MetMb). The formation of MetMb changes the color of meat from red to brown, thereby reducing its market value.1 Therefore, the preservation of bright red color of meat and meat products has a high degree of importance in food science and food industry.2 Polyphenols are well-known bioactive constituents of plants and plant-derived foods. They exhibit potent antioxidant activity, resulting in the prevention of the oxidative deterioration of food constituents. We attempted to use such polyphenols to prevent the oxidation of MbO2. However, the potently active polyphenols accelerated the oxidation of MbO2 to MetMb.3 The oxidation process was accelerated owing to the pro-oxidant nature of the quinone derivatives of these polyphenols. It should be noted that the reduction of the brown-colored MetMb to red-colored MbO2 is an alternative approach for conserving the fresh appearance of meat.4 Ascorbic acid is an extremely strong bioreductant that can reduce MetMb to MbO2.5 Some polyphenols also have strong reducing properties in addition to their potent antioxidant activities.6,7 Recently, the MetMb-reducing activities of various polyphenolic compounds was assessed.8 The results showed © 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%), potassium hexacyanoferrate(III), sodium hydrosulfite, and L-cysteine were obtained from Nacalai Tesque (Kyoto, Japan). Caffeic acid (purity >98%), ferulic acid (purity >98%), morin (purity >98% as hydrate), 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 Received: Revised: Accepted: Published: 9472

June 26, 2014 September 15, 2014 September 15, 2014 September 15, 2014 dx.doi.org/10.1021/jf5039508 | J. Agric. Food Chem. 2014, 62, 9472−9478

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>97%), chlorogenic acid (purity >95%), nordihydroguaiaretic acid (purity >97%), syringic acid (purity >95%), dihydrocaffeic acid (purity >98%), and tyrosinase (from mushroom) were obtained from SigmaAldrich (St. Louis, MO). Vanillic acid (purity >96%) was purchased from Wako Pure Chemicals (Osaka, Japan). Kaempferol (purity >97%), taxifolin (purity >96%), and 5,5′-dithiobis(2-nitrobenzoic acid) were obtained from Funakoshi (Tokyo, Japan). All organic solvents (extra pure grade or HPLC grade) were obtained from Nacalai Tesque. Distilled water produced by a water-distilling instrument (SA-2100E, EYELA, Tokyo, Japan) was used for all experiments. NMR spectra were obtained from an ECS-400 spectrometer (JEOL, Tokyo, Japan) using the manufacturer’s pulse sequences. High-resolution mass spectrometric data were obtained using a XEVO QtofMS spectrometer (Waters Japan, Tokyo, Japan) in negative ESI mode. A PU-2089 low-pressure gradient system (JASCO, Tokyo, Japan) equipped with an MD-2018 photodiode array detector (PDA) was employed for analytical HPLC. PDA data were analyzed using ChromNAV ver. 1.8 (JASCO). An LC-6AD system (Shimadzu, Kyoto, Japan) equipped with a UV-970 detector (JASCO) was used for preparative HPLC. Preparation of MetMb. The MetMb solution was prepared from a stock solution of MbO2, which was prepared using a method reported previously.8 Potassium hexacyanoferrate(III) (2 mg) was added to a solution of MbO2 (120 μmol/L) in a phosphate buffer (50 mmol/L, pH 7.4, 3 mL) at 23 °C. After gentle stirring for 0.5 min, the solution was desalted three times at 14 000g 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 (≈0.5 mL) was diluted with buffer, yielding 120 μmol/L of the MetMb solution (3 mL). The purity and concentration of the MetMb solution were confirmed using a UVmini 1240 UV/vis spectrometer (Shimadzu).12 Measurement of MetMb Reduction to MbO2. Briefly, 50 mmol/L of phosphate buffer (pH 7.4, 100 μL), 120 μmol/L of MetMb (150 μL, in the same buffer), test samples (polyphenols) dissolved in DMSO (18 or 9 mmol/L, 10 μL), and cysteine dissolved in water (4.5 or 2.3 mmol/L, 40 μL) were subsequently added to a 96-well microplate. The microplate was then incubated at 37 °C in a Multiskan GO microplate reader (Thermo Fisher Scientific, Yokohama, Japan) for 2 h, and absorbance was measured at 500 and 582 nm. The MbO2 and MetMb concentrations in the wells were calculated using the following equation8 MbO2 (μmol/L) = 89.7A582 − 32.9A500, where A500 and A582 refer to absorbance at 500 and 582 nm, respectively. The reductive conversion efficiency from MetMb to MbO2 was expressed as a percentage by calculating the MbO2 concentration relative to the initial concentration (60 μmol/L) of MetMb. HPLC and LC−MS Analyses of Caffeic Acid during MetMb Reduction. For caffeic acid analysis, 10 μL of caffeic acid in DMSO (18 mmol/L), 40 μL of cysteine in water (4.5 mmol/L), and 120 μL of MetMb buffer solution were added to 150 μL of a phosphate buffer (50 mmol/L, pH 7.4) in a microplate well. After shaking the plate, it was incubated at 37 °C in a Multiskan GO microplate reader. At 1 h intervals, an aliquot (5 μL) was removed from the above-mentioned reaction, and the reaction products were analyzed under the following conditions: column, a 250 mm × 4.6 mm i.d., 5 μm, Cosmosil 5C18AR-II (Nacalai Tesque); flow rate, 1.0 mL/min; solvent A, 1% acetic acid in water; solvent B, acetonitrile; gradient conditions: linear gradient of 5−100% solvent B (40 min); detection, absorbance at 280 nm. The concentrations of the caffeic acid and its derivative in the reaction mixture were measured by an external standard method and calculated using calibration curves, which were made from the peak areas of several concentrations of authentic samples. The MS data for each product were obtained by performing LC−MS. An additional aliquot (5 μL) was also withdrawn after 2 h for analyzing the compounds of peaks 1 and 2, and the analyses were carried out under the following conditions: column, a 250 mm × 4.6 mm i.d., 5 μm, Cosmosil 5C18-AR-II (Nacalai Tesque); flow rate, 0.5 mL/min; solvent A, 1% acetic acid in ultrapure water (Wako Pure Chemicals), solvent B, acetonitrile (LC−MS grade) (Merck, Darmstadt, Germany); gradient conditions, linear gradient of 5−100% solvent B

(80 min). 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; 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 by using MassLynx software v. 4.1 (Waters). Quantitative Analysis of Cysteine and Cystine during MetMb Reduction. Quantitation of cysteine in the reaction was carried out by using a modified Ellman’s method.13,14 Briefly, at 1 h intervals, an aliquot (20 μL) was withdrawn from the abovementioned reaction mixture. To the aliquot were subsequently added 30 μL of phosphate buffer (50 mmol/L, pH 7.4), 200 μL of distilled water, and 50 μL of 5,5′-dithiobis(2-nitrobenzoic acid) (0.4 mmol/L). After stirring for 5 min at 23 °C, 10 μL of the solution was withdrawn and the generated 5-hydrosulfuryl-2-nitrobenzoic acid was analyzed via HPLC under the following conditions: column, a 150 mm × 3.0 mm i.d., 3 μm, Cosmosil 3C18-EB (Nacalai Tesque); flow rate, 0.3 mL/min; solvent A, 0.1% phosphoric acid in water; solvent B, acetonitrile; gradient conditions, linear gradient of 0−100% of solvent B (30 min), detection at 330 min, with a retention time of 10.5 min. The quantitation of the cysteine in the incubating solution was performed using a calibration curve obtained by analyzing the reaction using known concentrations of cysteine. The quantitation of cystine was carried out by using the method of Mizuta and Sakai15 with a slight modification.16 Briefly, 2 or 3 h after the start of incubation, an additional aliquot (20 μL) was withdrawn from the reaction mixture, and 10 μL of benzoyl chloride (8 mmol/L in CH3CN) and 10 μL of triethylamine (8 mmol/L in CH3CN) were added to the aliquot at 0 °C with stirring. After keeping the mixture for 1 h at 0 °C, 10 μL of the reaction mixture was analyzed by HPLC under the following conditions: column, a 250 mm × 4.6 mm i.d., 5 μm, Cosmosil 5C18-AR-II (Nacalai Tesque); flow rate, 0.5 mL/min; solvent A, 0.1% trifluoroacetic acid in water; solvent B, acetonitrile; gradient conditions, linear gradient of 10−100% of solvent B (50 min); detection, absorbance at 254 nm. The concentration of cystine was determined using a calibration curve, which was derived from the analytical data of authentic cystine. Preparation of Cysteinylcaffeic Acids. 2′- and 5′-S-cysteinylcaffeic acids were synthesized enzymatically. Tyrosinase (2 mg) was added to a mixture of caffeic acid (50 mg) and cysteine (68 mg) in 10 mL of a phosphate buffer (50 mmol/L, pH 6.8). After keeping the mixture for 0.5 h at 23 °C, the mixture was concentrated to ≈10 mL in vacuo. The solution was then purified by a preparative HPLC as follows: column, a 250 × 20 mm i.d., 5 μm, Cosmosil 5C18-AR-II; solvent, CH3OH−1% acetic acid in H2O (15:85, v/v); flow rate, 8.0 mL/min; detection, 280 nm. 5′-S-Cysteinylcaffeic acid (5) (7 mg) and 2′-S-cysteinylcaffeic acid (4) (38 mg) showing peaks at the retention times of 33 and 48 min, respectively, were collected. 5′-S-Cysteinylcaffeic Acid. HR-ESIMS (m/z) [M − H]− calcd for C12H12NO6S 298.0385, found 298.0374; 1H NMR (DMSO-d6) δ 8.30 (1H, brs), 7.25 (1H, d, J = 15.6 Hz), 6.95 (1H, d, J = 1.6 Hz), 6.83 (1H, d, J = 1.6 Hz), 5.88 (1H, d, J = 15.6 Hz), 2.95 (1H, dd, J = 14.8 and 4.0 Hz), 2.90−2.75 (2H, m). 2′-S-Cysteinylcaffeic Acid. HR-ESIMS (m/z) [M − H]− calcd for C12H12NO6S 298.0385, found 298.0397; 1H NMR (DMSO-d6) δ 8.17 (1H, d, J = 15.8 Hz), 7.18 (1H, d, J = 8.2 Hz), 6.76 (1H, d, J = 8.2 Hz), 6.25 (1H, d, J = 15.8 Hz), 2.95 (1H, t, J = 7.8 Hz), 2.77 (2H, d, J = 7.8 Hz). Statistical Analysis. Data obtained from the experiments in triplicate are presented as mean ± standard deviation (SD). The differences between means were evaluated using the Student’s t-test. Statistical significance was considered at p < 0.05.

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RESULTS AND DISCUSSION

MetMb Reduction by Polyphenols in the Presence of Cysteine. In the previous investigation, the effect of various kinds of polyphenols and the related phytophenols on MetMb reduction was examined (Figure 1). Most of the polyphenols 9473

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Figure 1. Chemical structures of the polyphenols and related phenols investigated for MetMb reduction in the presence of cysteine and two synthesized cysteinylcaffeic acids.

cysteine. Caffeic acid, dihydrocaffeic acid, and hydroxytyrosol are powerful antioxidants17−19 and also have a strong reducing ability.20,21 Both activities are dependent on their catechol substructures. The catechol structure is readily oxidized to orthoquinone, which may interact with MbO2 to stimulate its oxidation to MetMb. When cysteine is present, the nucleophilic thiol group reacts with quinone to afford more stable polyphenolic derivatives as a result. Further investigation for elucidating the mechanism was carried out using the highly potent caffeic acid. Concentration Effect of Caffeic Acid and Cysteine on the Reduction of MetMb. Two concentration levels, 600 or 300 μmol/L, of polyphenols and cysteine for carrying out the experiment on MetMb reduction were employed, according to the method reported in a previous study.8 These abovementioned concentrations of polyphenols and cysteine are

did not show sufficient reducing activity for MetMb, as compared to ascorbic acid. Although cysteine shows no reducing activity toward MetMb, it was expected that cysteine would facilitate the polyphenol-mediated MetMb reduction. Figure 2 shows the results of MetMb reduction in air and the polyphenol-mediated MbO2 formation (concentrations of polyphenols 600 or 300 μmol/L) in the presence or absence of cysteine (600 or 300 μmol/L). The data are expressed as a percentage of the ratio of MbO2 produced in air to the total amount of myoglobins (MetMb + MbO2), which was calculated on the basis of the previously reported equation.8 The figure shows that the reducing activity of most polyphenols was remarkably enhanced by the presence of cysteine, whereas the activity of cysteine itself was extremely weak. In particular, the activity of caffeic acid (1), dihydrocaffeic acid (2), and hydroxytyrosol (3) increased significantly in the presence of 9474



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Figure 2. Effects of phenols (600 or 300 μmol/L) on MetMb reduction in the presence of cysteine (600 or 300 μmol/L). Results are expressed as mean ± standard deviation (SD) (n = 3) and * signifies significantly different data against data from the corresponding experiment in the absence of cysteine (t-test, p < 0.05).

much higher than the concentration of MetMb (60 μmol/L). Therefore, we initially examined the concentration effects of not only caffeic acid but also cysteine on the MetMb-reducing reaction, using a microplate method. The obtained matrix data are shown in Figure 3. Pure caffeic acid and cysteine

HPLC Analysis of MetMb Reduction in the Presence of Caffeic Acid and Cysteine and Identification of the Reaction Products. We carried out the HPLC analysis of the MetMb-reducing reaction of caffeic acid (600 μmol/L) in the presence of cysteine (600 μmol/L). In the HPLC profile, 2 h after the initiation of reaction (Figure 4), two noticeable

Figure 3. Matrix data of the various concentrations of caffeic acid and cysteine used for MetMb reduction. Reducing activity of MetMb is expressed as a percent of the formed MbO2, from the MetMb in the air. All data were presented as mean values of two independent experiments.

Figure 4. HPLC analytical profile for the reaction products from caffeic acid in the presence of cysteine, during the reducing reaction of MetMb at 2 h.

independently showed a concentration-dependent activity up to 2400 μmol/L, but these activities were not very significant. On the other hand, the mixtures of caffeic acid and cysteine showed a remarkable reducing activity. The optimal concentrations of caffeic acid and cysteine were between 600 and 2400 μmol/L for caffeic acid and 600 μmol/L for cysteine, 2 h after the reaction commenced. In general, higher concentrations of active compounds are expected to result in an increased efficiency. However, in this case, higher concentrations of cysteine did not show the expected linear concentrationdependent effect. Tang et al.22 have reported that glutathione, a biothiol related to cysteine, accelerated the oxidation of MbO2 to MetMb at pH 7.2 and 37 °C. This could be the reason why a higher concentration of cysteine (>600 μmol/L) prevented MbO2 formation.

product peaks were observed at the retention times of 7.6 and 8.1 min, respectively, in addition to a large peak corresponding to caffeic acid (10.7 min). To obtain the structure information corresponding to these two peak compounds, they were subjected to MS analysis using LC−TOFMS. High-resolution MS data for peak 1 (m/z 298.0384 [M − H]−) and peak 2 (m/ z 298.0400 [M − H]−) indicated that the molecular formula of both peak compounds was C12H13NO6S. From the molecular formula, peaks 1 and 2 were thought to be the isomeric coupling products of an equimolar amount of caffeic acid and cysteine. In our previous study,10 the radical oxidation of caffeic ester in the presence of a cysteine derivative afforded 2′-Ssubstituted caffeic ester as the main product, which was produced by the addition of cysteinyl thiol to a quinone derivative of the caffeic ester. Therefore, we prepared a similar 9475



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same concentration of cysteine and caffeic acid without MetMb, and the corresponding results are shown in Figure 5B. Figure 5B shows that the reaction rate of cysteine in the absence of MetMb was lower than that in the presence of MetMb. Moreover, caffeic acid did not decrease during the same time period. These results indicated that MetMb primarily oxidized caffeic acid and then produced two cysteinylcaffeic acids from a subsequent coupling reaction of the remaining cysteine and the oxidized caffeic acid (quinone of caffeic acid). In our previous study,3 high concentrations of caffeic acid were found to promote the oxidation of MbO2 to MetMb. This MbO2 oxidation may be attributed to interaction between MbO2 and the electrophilically oxidized polyphenols such as caffeic quinone.24,25 When cysteine was present in the solution, the caffeic quinone that was derived by the oxidation of caffeic acid quickly reacted with the cysteine to form cysteinylcaffeic acids. The cysteinylcaffeic acids might not stimulate MbO2 oxidation because of the absence of any reactive quinone structures. Effect of Cysteinylcaffeic Acid on the Redox States of Mb. During the reduction of MetMb, caffeic acid was oxidized to a corresponding quinone derivative. However, the coexisting cysteine quenched the quinone to produce cysteinylcaffeic acids. Next, we examined the redox activities of the cysteinylcaffeic acids on Mb. Cysteinylcaffeic acids were synthesized with tyrosinase oxidation, and the major product obtained (2′-S-cysteinylcaffeic acid) was used for this assessment. The MetMb-reducing activity and MbO2-oxidizing activity of 2′-S-cysteinylcaffeic acid were measured. The corresponding results are shown in parts A and B of Figure 6, respectively. Figure 6A shows that 2′-S-cysteinylcaffeic acid

quinone derivative of caffeic acid and then added cysteine to the quinone. A quinone of caffeic acid was synthesized with the help of tyrosinase oxidation, and cysteine was then reacted to obtain the two products, whose structures were fully determined as 2′-S-cysteinylcaffeic acid (a major product) and 5′-S-cysteinylcaffeic acid (a minor product) by performing MS and NMR. These synthetically obtained cysteinylcaffeic acids were identical to the peak compounds by comparison of the retention times of the HPLC and MS data. Therefore, the structures of the compounds giving peaks 1 and 2 were determined to be 5′-S-cysteinylcaffeic acid (5) and 2′-Scysteinylcaffeic acid (4), respectively. (Figure 1) Time Course Analysis of Caffeic Acid and Cysteine and the Oxidation Products during MetMb Reduction. The time-course analytical results of the formation of two cysteinylcaffeic acids (2′-S-cysteinylcaffeic acid and 5′-Scysteinylcaffeic acid) and the decrease of caffeic acid and cysteine in the MetMb-reducing solution are summarized in Figure 5A−C. Figure 5C shows that both the coupling products

Figure 5. Time-course analytical data for MbO2 (A); caffeic acid, cysteine, and cystine (2 or 3 h data only) (B); and reaction products (C) in the reducing reaction of MetMb. All the analytical experiments were performed in duplicate, and the data are expressed in terms of the corresponding mean values. Figure 6. Concentration effect of 2′-S-cysteinylcaffeic acid on MetMb reduction (A and C) and MbO2 oxidation (B and D) at 37 and 25 °C, respectively. Redox values of MetMb and MbO2 were expressed as the MbO2 percent of Mbs in the reaction. All the analytical experiments were performed in duplicate, and the data are expressed as mean values.

(2′-S-cysteinylcaffeic acid and 5′-S-cysteinylcaffeic acid) increased during the first 2 h. During this period, MbO2 was continuously accumulated from the reduction of MetMb, as shown in Figure 5A. The amount of caffeic acid was partially decreased at 2 h, as shown in Figure 5B. Figure 5B also shows that cysteine decreased drastically during this time period. This decreased cysteine was oxidatively converted to cystine owing to the corresponding cystine concentration measured in the 2 h reaction. During the MetMb-reducing reaction, cysteine was completely oxidized. However, Romero et al.23 reported that the reducing activity of cysteine toward MetMb was negligible. It should be noted that cysteine was unstable under the alkaline aerobic conditions. Therefore, we measured reaction rate of the

exhibited a potent MetMb-reducing activity in a concentrationdependent manner, but the same concentrations of 2′-Scysteinylcaffeic acid did not show any oxidation activity of MbO2 as compared to the control experiment, as shown in Figure 6B. These results were also observed clearly at lower temperature conditions corresponding to 25 °C as an ambient temperature (Figure 6C,D). At that temperature, MbO2 was 9476



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stable until 5 h, and 2′-S-cysteinylcaffeic acid did not affect the stability of MbO2 during this time period (Figure 6D). However, 2′-S-cysteinylcaffeic acid had a slower but more effective reducing activity for the conversion of MetMb to MbO2, as shown in Figure 6C. It is known that sulfur substitutions in polyphenols alter their redox properties.10,26 Substituted cysteinyl sulfur can modulate the pro-oxidant activity of caffeic acid and make it nonreactive toward MbO2, while still retaining the powerful reducing activity of the caffeic acid. One of the objectives of the current study3,8 was the development of a polyphenol-based technology for the preservation of fresh meat color. The cysteine-modified polyphenols may thus provide a novel alternative for the prevention of changes of meat color. From the results obtained in this study, some highly antioxidative polyphenols produced bright red MbO2 in the presence of cysteine by reducing brown MetMb at pH 7.4. The mechanism is thought to be a combination of the potent reducing activity of polyphenols and the preventive effect of cysteine on the expression of the pro-oxidant property of polyphenols by nucleophilic addition of cysteinyl thiol to oxidized polyphenols, as illustrated in Figure 7. Cysteinylcaffeic

REFERENCES

(1) Issanchu, S. Consumer expectations and perceptions of meat and meat product quality. Meat Sci. 1999, 43, S5−19. (2) Mancini, R. A.; Hunt, M. C. Current research in meat color. Meat Sci. 2005, 71, 100−121. (3) Masuda, T.; Inai, M.; Miura, Y.; Masuda, A.; Yamauchi, S. Effect of polyphenols on oxymyoglobin oxidation: Prooxidant activity of polyphenols in vitro and inhibition by amino acid. J. Agric. Food Chem. 2013, 61, 1097−1104. (4) Bekhit, A. E. D.; Faustman, C. Metmyoglobin reducing activity. Meat Sci. 2005, 71, 407−439. (5) Tukahara, K.; Yamamoto, Y. Kinetic studies on the reduction of metmyoglobin by ascorbic acid. J. Biochem. 1983, 93, 15−22. (6) Lien, E. J.; Ren, S.; Bui, H.-H.; Wang, R. Quantitative structure− activity relationships analysis of phenolic antioxidants. Free Radical Biol. Med. 1999, 26, 285−294. (7) Pietta, P.-G. Flavonoids as antioxidants. J. Nat. Prod. 2000, 63, 1035−1042. (8) Inai, M.; Miura, Y.; Honda, S.; Masuda, A.; Masuda, T. Metmyoglobin reduction by polyphenols and mechanism of the conversion of metmyoglobin to oxymyoglobin by quercetin. J. Agric. Food Chem. 2014, 62, 893−901. (9) Khalife, H. K.; Lupidi, G. Reduction of hypervalent states of myoglobin and hemoglobin to their ferrous forms by thymoquinone: The role of GSH, NADH and NADPH. Biochim. Biophys. Acta 2008, 1780, 627−637. (10) Fujimoto, A.; Inai, M.; Masuda, T. Chemical evidence for the synergistic effect of a cysteinyl thiol on the antioxidant activity of caffeic and dihydrocaffeic esters. Food Chem. 2013, 138, 1483−1492. (11) Masuda, T.; Miura, Y.; Inai, M.; Masuda, A. Enhancing effect of a cysteinyl thiol on the antioxidant activity of flavonoids and identification of antioxidative thiol-adducts of myricetin. Biosci. Biotechnol. Biochem. 2013, 77, 1753−1758. (12) Bowen, W. J. The absorption spectra and extinction coefficients of myoglobin. J. Biol. Chem. 1949, 179, 235−245. (13) Ellman, G. L. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 1959, 82, 70−77. (14) Sedlak, J.; Lindsay, R. H. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman’s reagent. Anal. Biochem. 1968, 25, 192−205. (15) Mizuta, H.; Sakai, J. High performance liquid chromatographic determination of cysteine esters. Jpn. Soc. Anal. Chem. 1978, 27, 744− 748. (16) Miura, Y.; Honda, S.; Masuda, A.; Masuda, T. Antioxidant activities of cysteine derivatives against lipid oxidation in anhydrous media. Biosci. Biotechnol. Biochem. 2014, 78, 1452−1455. (17) Gülçin, I.̇ Antioxidant activity of caffeic acid (3,4-dihydroxycinnamic acid). Toxicology 2006, 217, 213−220. (18) Silva, F. A. M.; Borges, F.; Guimarăes, C.; Lima, J. L. F. C.; Matos, C.; Reis, S. Phenolic acids and derivatives: Studies on the relationship among structure, radical scavenging activity, and physicochemical parameters. J. Agric. Food Chem. 2000, 48, 2122− 2126. (19) Papadopiulos, G.; Boskou, D. Antioxidant effect of natural phenols on olive oil. J. Am. Oil Chem. Soc. 1991, 68, 669−671. (20) Nenadis, N.; Boyle, S.; Bakalbassis, E. G.; Tsimidou, M. An experimental approach to structure−activity relationships of caffeic and dihydrocaffeic acids and related monophenols. J. Am. Oil Chem. Soc. 2003, 80, 451−458. (21) Carrasco-Pancorbo, A.; Cerretani, L.; Bendini, A.; SeguraCarretero, A.; Del Carlo, M.; Gallina-Toschi, T.; Lercker, G.; Compagnone, D.; Fernándes-Gutiérrez, A. Evaluation of the antioxidant capacity of individual phenolic compounds in virgin olive oil. J. Agric. Food Chem. 2005, 53, 8918−8925. (22) Tang, J.; Faustman, C.; Lee, S.; Hoagland, T. A. Effect of glutathione on oxymyoglobin oxidation. J. Agric. Food Chem. 2003, 51, 1691−1695. (23) Romero, F. J.; Ordonez, I.; Arduini, A.; Cadenas, E. The reactivity of thiols and disulfides with different redox states of

Figure 7. Proposed mechanism for redox change between MetMb and MbO2 by polyphenols in the presence of thiols.

acid, a thiol-substituted polyphenol, exhibited no oxidationstimulating property toward MbO2 after the reduction of MetMb. These results show that the polyphenols were indeed useful in retaining the bright red color of MbO2, with support from biothiols such as cysteine. The bright red color of meat is very important in the food industry. These findings in this investigation should contribute not only to meat science but also to the chemistry of meat products.

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AUTHOR INFORMATION

Corresponding Author

*Fax: +81-88-656-7244. E-mail: [email protected]. Funding

Financial support from The Ito Foundation (Meguro, Tokyo, Japan) is gratefully acknowledged. Notes

The authors declare no competing financial interest. 9477



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myoglobin. Redox and addition reaction and formation of thiyl radical intermediates. J. Biol. Chem. 1992, 267, 1680−1688. (24) Alderton, A. L.; Faustman, C.; Liebler, D. C.; Hill, D. W. Induction of redox instability of bovine myoglobin by addition with 4hydroxy-2-nonenal. Biochemistry 2003, 42, 4398−4405. (25) Hurrell, R. F.; Finot, P. A. Protein−polyphenol reactions 1. Nutritional and metabolic consequences of the reaction between oxidized caffeic acid and the lysine residue of casein. Br. J. Nutr. 1982, 47, 191−211. (26) Malmström, J.; Jonsson, M.; Cotreave, I. A.; Hammarström, L.; Sjödin, M. The antioxidant profile of 2,3-dihydrobenzo[b]furan-5-ol and its 1-thio, 1-seleno, amd 1-telluro analogues. J. Am. Chem. Soc. 2001, 123, 3434−3440.

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Reducing Effects of Polyphenols on Metmyoglobin and the in Vitro

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