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  • 1 University of Life Sciences, Skromna 8, 20-704 Lublin, Poland
  • 2 Institute of Soil Science and Plant Cultivation, State Research Institute, Krancowa 8, 24-100 Pulawy, Poland
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The use of polyphenols in food fortification is a common custom generally carried out to increase its nutritional value. In this paper, ground chili pepper was proposed as a potential functional coffee additive. Various phenolic compounds present in this spice were analyzed by a new, sensitive, and selective ultrahigh-performance liquid chromatography combined with mass spectrometry (UPLC–MS). Separation was done on a column filled with a modified silica gel RP-18, in gradient solvent systems A (1% H3PO4 in water) and B (40% CH3CN in solution A). The capsaicin was found as the main phenolic compound of ground chilli pepper, which concentration was 295.95 mg g−1. It was demonstrated that quercetin is present in this spice also in different forms: not only as dihydrocapsaicin but also as quercetin-3-O-deoxyhexoside-glucuronide and quercetin-3-O-deoxyhexoside, whereas luteolin in the form of three compounds: luteolin-7-O-dihexoside, luteolin-6-C-hexoside-8-C-pentoside, and luteolin-7-O-malonyl-dihexosyl-pentoside. We have also identified apigenin-6-C-hexoside-8-C-pentoside. Furthermore, this paper, for the first time, evaluates the potential bioaccessibility of and interactions between compounds with multidirectional antioxidant properties from coffee and ground chili pepper. All samples, coffee, chili, and a mixture of the two showed ability to scavenge free radicals and chelate iron ions and were characterized by reducing power. The level of these activities changed after simulated gastrointestinal digestion. In the mixtures of water extracts, phytochemicals acted synergistically in the case of five from six tested methods. Interestingly, after digestion, in vitro chili extract lost ability to scavenge O2 radicals; that is why it was impossible to determine the interactions between coffee and chili in this case. Moreover, an antagonism in the action was observed for those cases, where, in water extracts, we have identified synergistic interaction.

Abstract

The use of polyphenols in food fortification is a common custom generally carried out to increase its nutritional value. In this paper, ground chili pepper was proposed as a potential functional coffee additive. Various phenolic compounds present in this spice were analyzed by a new, sensitive, and selective ultrahigh-performance liquid chromatography combined with mass spectrometry (UPLC–MS). Separation was done on a column filled with a modified silica gel RP-18, in gradient solvent systems A (1% H3PO4 in water) and B (40% CH3CN in solution A). The capsaicin was found as the main phenolic compound of ground chilli pepper, which concentration was 295.95 mg g−1. It was demonstrated that quercetin is present in this spice also in different forms: not only as dihydrocapsaicin but also as quercetin-3-O-deoxyhexoside-glucuronide and quercetin-3-O-deoxyhexoside, whereas luteolin in the form of three compounds: luteolin-7-O-dihexoside, luteolin-6-C-hexoside-8-C-pentoside, and luteolin-7-O-malonyl-dihexosyl-pentoside. We have also identified apigenin-6-C-hexoside-8-C-pentoside. Furthermore, this paper, for the first time, evaluates the potential bioaccessibility of and interactions between compounds with multidirectional antioxidant properties from coffee and ground chili pepper. All samples, coffee, chili, and a mixture of the two showed ability to scavenge free radicals and chelate iron ions and were characterized by reducing power. The level of these activities changed after simulated gastrointestinal digestion. In the mixtures of water extracts, phytochemicals acted synergistically in the case of five from six tested methods. Interestingly, after digestion, in vitro chili extract lost ability to scavenge O2 radicals; that is why it was impossible to determine the interactions between coffee and chili in this case. Moreover, an antagonism in the action was observed for those cases, where, in water extracts, we have identified synergistic interaction.

Introduction

Phenolic compounds have began to attract the interest of researchers, because they show promise of being powerful antioxidants that can protect the human body from free radicals, the formation of which is associated with the normal natural metabolism of aerobic cells [1].

Most people start their day tasting a cup of coffee, which is the most popular beverage in the world. Many previous studies have reported adverse effects of coffee consumption (e.g., anemia, hypertension, depression, osteoporosis) [2]. Nowadays, epidemiological and experimental studies have shown many positive effects of regular coffee intake on neurological conditions, psychoactive responses, and metabolic disorders, as well as positive effect of coffee on gonad and liver function [3]. It has been also proven that low caffeine doses (60–200 mg/day, usually consumed during the day) enhance alertness and increase perception and levels of concentration on simple tasks. These properties are assigned to the presence of bioactive compounds with strong antioxidant and radical scavenging activities, especially polyphenols [4].

Since ancient times, spices and herbs have been added to foods as folk medicines, food preservatives, and flavoring agents. Presently, there is an increasing interest in spices and aromatic herbs, both in industry and in scientific research [5]. It is worth noting that many herbs and spices usually used to flavor dishes contain phenolic compounds which have been reported to show good antioxidant activity [6]. The powdered chili pepper used in this study as a coffee supplement affects the beverage antioxidant properties, because this spice is a source of bioactive compounds. Hot chili pepper belongs to the Solanaceae family and is one of the most popular spices in the world, which is appreciated for its sensory attributes of color, pungency, and aroma [7], as well as its wide array of important nutritional antioxidants [8]. However, there is no information on the antioxidant property of coffee with ground chili pepper as a supplement and its potential for the management of oxidative stress-related metabolic disorders.

Few published papers have reported the determination of major phenolics in pepper fruit Capsicum annuum L. using analytical methods, including liquid chromatography (LC) [9], high-performance liquid chromatography (HPLC) [10], and ultrahigh-performance liquid chromatography with diode array detection (UPLC–DAD) in leaves of a wild C. annuum [11]. However, HPLC method suffered from low resolution and sensitivity, long run time, and using a few analytes.

In this study, phenolic compounds were analyzed by a sensitive and selective UPLC–MS method in hot water extracts of chili (as a coffee supplement) and their antioxidant properties were determined. Besides that, the aim of this study was to test two hypotheses: (1) interactions between bioactive compounds play a crucial role in the creation of the nutraceutical potential of coffee supplemented with chili; and (2) conditions during simulated gastrointestinal digestion cause significant differences in relationships between the phenolic compounds of analyzed material.

Experimental

Plant Material and Chemical Reagents.

The research material accounted for ground coffee, typical average quality, available on the Polish market. Chili pepper was bought in the form of ground spice from a local market (Lublin, Poland). Analytical grade reagents were purchased from Sigma-Aldrich Company (Poznan, Poland) and J.T. Baker (Phillipsburg, NJ).

Sample Preparation.

The water-soluble antioxidants were extracted by pouring over 500 mg of coffee and chili with 30 mL of boiling water. Then, the samples were shaken for 30 min at 37 °C. After centrifugation (15 min, 20 °C, 8000g), the supernatants were decanted, and the final volume was brought to 50 mL with distilled water. The water (raw) extracts concentration was 10 mg mL−1 dry weight (DW).

In Vitro Digestion.

In vitro digestion was carried out according to the method described by Gawlik-Dziki [12], with slight modification. For gastric digestion, 300 U mL−1 of pepsin A in 0.03 M HCl (pH 1.2) was used. After that, samples were adjusted to pH 6 with 0.1 M NaHCO3, and intestinal digestion was conducted (0.05 g of pancreatin and 0.3 g of bile extract in 35 mL 0.1 M NaHCO3). Finally, 5 mL of 120 mM NaCl and 5 mM KCl was added to the samples. The final concentration of the gastrointestinally digested extract was 3.6 mg mL−1 of dry weight (DW).

Analytical Procedures.

Qualitative and quantitative analysis of phenolic compounds were analyzed using a Waters ACQUITY UPLC™ system (Waters Corp., Milford, MA, USA), consisting of a sample manager, binary pump system, column manager, and PDA detector (also from Waters Corp.). Separation was done on a column filled with a modified silica gel RP-18 (Vertex Eurosil Bioselect 300 Å, Ø 5 μm, 4 × 30 mm, endcapped), in gradient solvent systems A (1% H3PO4 in water) and B (40% CH3CN in solution A), in such a proportion that the CH3CN concentration was 8% in 0 to 10 min; 20% in the 40th min; and 40% in the 55th min; the flow speed was 1 mL min−1, and the detection was at 225 nm. Particular derivatives of flavonoids and phenolic acids were quantitatively determined with the use as standards phenolic compounds and identified by spectral methods such as MS. Quantitative determination of the capsaicinoids fraction in chili pepper was conducted by the UPLC method in an isocratic system with 55% CH3CN, with the flow speed of 1 mL min−1 and detection at 255 nm. The capsaicin and dihydrocapsaicin content was determined on the basis of a standard solution of capsaicin containing also dihydrocapsaicin (Merck).

Analysis of Antioxidant Activities.

Antioxidant activities (except reducing power, RED) were expressed as EC50 (extract concentration): the amount of sample (mg DW) needed to obtain 50% activity per 1.0 mL of the initial solution. Reducing power determined as EC50 is the effective concentration at which the absorbance was 0.5 for reducing power and was obtained by interpolation from linear regression analysis.

Free radical-scavenging activity was determined by the ABTS•+ method according to Re et al. [13]. The ABTS solution was prepared with potassium persulfate, diluted in ethanol, and left in the dark for 16 h to allow radical development. The solution was diluted to reach absorbance measures around 0.70–0.72 at 734 nm. 1.8 mL ABTS•+ solution was added to 0.04 mL of each sample. The absorbance was measured at 734 nm after 3 min of reaction. Deionized water was a blank sample. Percentage inhibition of the ABTS•+ radical was calculated as follows: scavenging % = [1 − (As/Ac)] × 100, where As is the absorbance of sample; Ac, absorbance of control (ABTS).

Chelating power was determined by the method of Guo et al. [14]. The samples (0.5 mL) were mixed with a 0.1 mL of 2 mM L−1 FeCl2 solution and 0.2 mL of 5 mM L−1 ferrozine and then left to stand at room temperature for 10 min. The absorbance was measured at 562 nm. The ability to inhibit ferrozine–Fe2+ complex formation was determined as follows: % inhibition = [1 − (AA/AC)] × 100, where AC is the absorbance of control and AA is the absorbance of sample.

Hydroxyl radicals (OH scavenging assay) were generated by Fenton reaction in the system of FeSO4 and H2O2. The reaction mixture consisted of 0.5 mL of FeSO4 (8 mM), 0.8 mL of H2O2 (6 mM), 0.5 mL distilled water, 1.0 mL of extract, and 0.2 mL sodium salicylate (20 mM). The total mixture (3.0 mL) was incubated for 1 h at 37 °C, and then, the absorbance was noted at 562 nm. The scavenging activity was calculated as follows: scavenging activity [%] = [1 − (A1A2)/A0] × 100, where A0 is the absorbance of the control (without extract); A1, absorbance of the extract addition; and A2, absorbance without sodium salicylate.

Reducing power (RED) was determined by the method of Oyaizu [15]. A 0.5 mL of sample was mixed with 0.5 mL (200 mM) of sodium phosphate buffer (pH 6.6) and 0.5 mL potassium ferricyanide (10 g L−1). The incubation of samples was carried out by 20 min at 50 °C. After that, 0.5 mL of TCA (100 g L−1) was added and mixtures were centrifuged at 650×g by 10 min. Upper layer (1 mL) of supernatant was mixed with 1 mL of distilled water and 0.2 mL of ferric chloride (1 g L−1). The absorbance was measured in the spectrophotometer at 700 nm.

Assay for superoxide dismutase-like activity (SOD-like) was assayed on the basis of the method described by Marklund and Marklund [16], with modification. Sample solution, Tris–HCl buffer, and 0.2 mL of 7.2 mM pyrogallol were the reaction mixture, which was kept at 25 °C for 10 min. The amount of oxidized pyrogallol was measured spectrophotometrically (420 nm) after stopping the reaction by HCl. The SOD-like activity was calculated using the following formula: SOD-like activity [%] = [1 − (AS/AC)] × 100, where AS is the sample absorbance; AC, absorbance of control sample.

To determine the superoxide anion scavenging activity (SASA), nitroblue tetrazolium (NBT) solution, 1 mL NADH, and 0.1 mg of sample solution were mixed. The reaction was initiated by adding 100 μL of phenazine methosulfate (PMS) solution to the reaction mixture. Samples were incubated for 5 min at 25 °C, and the absorbance at 560 nm was measured against blank samples. The decreased absorbance of the reaction mixture indicated an increased superoxide anion scavenging activity. We used the following equation to evaluate the affinity of test material to quench superoxide anion: SASA [%] = [(A(0)AA)/A(0)] × 100, where A(0) is the absorbance of control; AA, absorbance of sample.

Analysis of Interactions.

The coffee brew and the chili brew, the supplement, were analyzed separately and in volume combinations (4:1, 3:2, 1:1, 2:3, 1:4, v/v) coffee–chili. The interaction factor (IF), which provides an explanation for the mode of interaction, was also determined, according to Gawlik-Dziki [12]: IF = AM/AT, where AM is the activity of samples mixtures, and AT, theoretically calculated mixture activity (IF value < 1 synergistic interaction; IF > 1 antagonism; IF ≈ 1 additive reaction).

Statistical Analysis.

Each calibration curve was constructed by running standards of different concentrations, in triplicate. The experimental results were mean ± SD of three parallel experiments (n = 3). Statistical tests were carried out using STATISTICA 7.0 (StatSoft, Inc., Tulsa, USA) for mean comparison using Tukey's test. Statistical significance was declared at p ≤ 0.05.

Results and Discussion

Qualitative–Quantitative Analysis of Coffee and Chili Phenolic Compounds.

UPLC–MS analyses allowed the identification of 14 phenolic compounds in coffee extract such as caffeoyl quinic acid and its isomers, caffeoyl shikimic acid, feruloyl quinic acid, dicaffeoyl quinic acid, and caffeoyl-feruloylquinic acid [18]. Antioxidants of the hydroxycinnamic acids group, such as combined or conjugated forms of caffeic, chlorogenic, coumaric, ferulic, and sinapic acids, are also found in coffee beverage. In addition, other biologically active compounds found in coffee are nicotinic acid, trigonelline, pyrogallic acid, quinolinic acid, tannic acid, and caffeine [19]. The beverage is also known for the antioxidant properties of its components, especially caffeine, hydroxycinnamic acids, and melanoidins produced as a result of Maillard reaction [20]. Furthermore, we have identified eight phenolic compounds from the ground chili infusion (Figure 1). The capsaicin was found as the main phenolic compound, the concentration of which was 295.95 ± 13.00 mg g−1 DW. In the present work, it was shown that quercetin is present in dried ground red pepper fruits in different forms: not only as dihydrocapsaicin but also as quercetin-3-O-deoxyhexoside-glucuronide and quercetin-3-O-deoxyhexoside, whereas luteolin occurs in the form of three compounds: luteolin-7-O-dihexoside, luteolin-6-C-hexoside-8-C-pentoside, and luteolin-7-O-malonyl-dihexosyl-pentoside. We have also identified apigenin-6-C-hexoside-8-C-pentoside, the concentration of which was the lowest (11.78 ± 0.40 mg g−1 DW). It is worth noting, that the total concentration of chili phenolic compounds (966.27 ± 14.46 mg g−1 DW) was much higher than in the case of coffee extract (224.9 ± 11.25 mg g−1 DW) and extracts of different coffee supplement such as cinnamon (42.6 ± 2.11 mg g−1 DW) [17] and ginger (5.4 ± 0.01 mg g−1 DW) [18]. However, amount of bioactive constituents depends on the extraction procedures. The results obtained in these studies were not consistent with the findings of other authors. Shan et al. [21] stated the total phenolic content of methanolic chili extract as 0.86 ± 0.004 g of gallic acid equivalent (GAE/100 g DW), which is a much lower result than the one obtained by us for the aqueous extracts of ground chili pepper. An alternative to the consumption of fresh vegetables is their dried form, which allows using them during the off-season. However, food products are sensitive to drying temperatures and methods that can induce degradation and change the nutritional and functional properties of the products [22]. In their work, Ozgur et al. [22] proved that during drying, the amount of phenolic compounds decreased from 130.79 ± 2.14 g of GAE/100 g of DW to 89.82 ± 4.72 g of GAE/100 g of DW in red chili pepper, which means that, in comparison with these results, ground chili pepper used in our study was rich in phenolic compounds.

Figure 1.
Figure 1.

UPLC–UV chromatogram (255 nm) of chili extract and concentration of identified chili phenolic compound

Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2016.00173

Antioxidant Activity and Interactions of Bioactive Compounds.

Numerous in vitro assays can be used to determine the antioxidant potential of plant extracts. Antioxidant activities in this study were determined using six different methods presented in Figure 2. The antiradical capacity of the water-soluble compounds of coffee extract in combination with chili extract has been revealed by ABTS method. Antiradical power of coffee was much higher (EC50 = 2.07 mg mL−1) in comparison to chili extract (EC50 = 16.11 mg mL−1; Figure 2), in raw extracts. It was also found that, in both samples, the simulated gastrointestinal digestion caused a significant increase in antiradical power, while EC50 values decreased accordingly to 1.60 mg mL−1 for coffee and to 6.59 mg mL−1 for chili (Figure 2). As presented in Figure 3A, isobole took the concave form. These results indicate that antiradical scavengers included in coffee and chili acted synergistically prior digestion. Interesting results were obtained during the isobolographic analysis of the tested extracts subjected to the simulated digestion. The changes that have occurred in the extracts of coffee and chili caused that after the process of digestion in vitro, where the extracts acted antagonistically (Figure 4A). Examining changes in the interactions that occur between coffee and ginger [18], we have observed the same kind of interactions as in the case of coffee with chili presented in this paper. In examining the CHEL, the EC50 values were similar: 0.35 mg mL−1 for coffee extract and 0.39 mg mL−1 for chili extract. However, it is worth noting that, after simulated digestion process, EC50 values decreased significantly to 0.11 mg mL−1 for coffee and 0.21 mg mL−1 for chili extract (Figure 2), which means that the gastrointestinal system is a good extractor of compounds able to chelate transition metal ions. Furthermore, these compounds present in coffee and chili extracts acted antagonistically, prior to and after simulated digestion — both isoboles (Figures 3B and 4B) are convex. Moreover, the antagonism of water extracts IF = 1.12 (Table 1) was lower than after digestion IF = 1.43 (Table 2). Subsequently, the ability to OH radical neutralization was determined. Experience has shown that chilli water extract was characterized by the highest activity (Figure 2); hence, EC50 value was the lowest: 0.73 mg mL−1. Interesting results were obtained during the analysis of the tested extracts subjected to simulated digestion. Chili extract's ability to OH radical neutralization decreased to 1.93 mg mL−1, when the activity of the coffee digested extract increased from 2.53 to 0.61 mg mL−1. Taking into account the isobolographic analysis (Figures 3C and 4C), compounds capable of OH radical neutralization acted synergistically before digestion, but after this process, we have observed a reverse kind of interaction. In determining the ferric reducing antioxidant power, we have obtained very low EC50 value for coffee water extract (EC50 = 0.86 mg mL−1) but much higher for chili extract (5.83 mg mL−1). After digestion, EC50 value for coffee increased to 2.52 mg mL−1; however, for chili extract, we have observed a slight increase in activity recorded as a decrease in value of EC50 = 4.94 mg mL−1 (Figure 2). Despite the changes at EC50 values, bioactive compounds with the ferric reducing antioxidant power acted antagonistically prior to and after simulated digestion — both isoboles (Figures 3D and 4D) are convex. Taking into account raw extracts, SOD-like activity was much higher for coffee water extract (EC50 = 9.46 mg mL−1) in comparison to chili extract (EC50 = 25.16 mg mL−1; Figure 2). Moreover, after digestion, coffee capacity decreased significantly to EC50 value of 33.60 mg mL−1, and we have observed no activity for chili extract. That is why it was only possible to determine the interactions between water and nondigested extracts. Isobole (Figure 3E) is concave, which indicates a synergistic interaction. The next analysis was an evaluation of SASA. In this assay, we have observed activity only in the case of water extracts. EC50 value for coffee extract amounted to 6.17 mg mL−1, and in turn, superoxide anion scavenging activity of chili extract was much lower: EC50 = 21.55 mg mL−1 (Figure 2). Isobole (Figure 3F) is concave, which indicates a synergistic interaction. After digestion, both extracts lost their ability to superoxide anion neutralization. Oxidative stress in cells can result from an increase in the levels of reactive oxygen species (ROS). The results of our research indicate both the multidirectional activity of natural antioxidants and their mutual interactions. They also underline the necessity of determining the antioxidant activity through the use of diverse mechanisms. Pekkarinen et al. [23] stated that the antioxidant potential might vary as affected by the method of its determination, and within the same method, the differences in the polarity of the medium may also vary due to the interactions between the antioxidants and other chemical compounds that play a major role in their activity.

Figure 2.
Figure 2.

Comparison of antioxidant activities of coffee and chili extracts before and after in vitro digestion

Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2016.00173

Figure 3.
Figure 3.

Isobole curves for 50% activity of coffee and chili mixtures before digestion: A, ABTS radical scavenging activity; B, chelating power (CHEL); C, OH scavenging assay (OH); D, reducing power assay (RED); E, superoxide dismutase-like activity (SOD-like); F, superoxide anion scavenging activity (SASA)

Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2016.00173

Figure 4.
Figure 4.

Isobole curves for 50% activity of coffee and chili mixtures after digestion: A, ABTS radical scavenging activity; B, chelating power (CHEL); C, OH scavenging assay (OH); D, reducing power assay (RED)

Citation: Acta Chromatographica Acta Chromatographica 30, 1; 10.1556/1326.2016.00173

Table 1.

Comparison of interaction factors (IF) of mixtures of coffee with chili water extracts

SampleActivityAMaATbIF
Coffee–chili mixture (1:1, v/v)Antiradical potential3.419.090.38
Chelating power0.410.371.12
OH• scavenging assay0.931.630.57
Reducing power1.213.340.36
Superoxide dismutase-like activity12.3217.310.71
Superoxide anion scavenging activity8.4613.860.61

Measured activity (expressed as EC50 [mg mL−1]).

Theoretical calculated activity (expressed as EC50 [mg mL−1]).

The bioaccessibility of dietary polyphenols is principally relevant to their beneficial effects. In order to determine the potential bioaccessibility of compounds with antioxidative potential, we further analyzed the properties of raw coffee and chili extracts (before digestion in vitro) and those extracts subjected to simulated gastrointestinal processing. In turn, Saura-Calixto et al. [24] in their study stated that polyphenols from beverages are completely bioaccessible and they are the largest contributors of bioaccessible polyphenols in the small intestine because they pass directly into the intestinal fluids. Polyphenols associated with the indigestible fraction, a major part of the polyphenols bioaccessible in the small intestine may also reach the colon because of their low bioavailability. The method initially proposed for identification of interactions between active compounds was isobolographic analysis. As Tables 1 and 2 present, the isobole curves shown in Figures 3(A–F) and 4(A–D) are confirmed by the IF. We have observed significant changes in the kinds of interactions between extracts after in vitro digestion in comparison to water, in raw extracts. This demonstrates that interaction with the food matrix and/or changes during simulated gastrointestinal digestion caused significant differences in the relationships between phenolic compounds present in coffee and chili extracts.

Table 2.

Comparison of interaction factors (IF) of mixtures of coffee with chili digested extracts

SampleActivityAMaATbIF
Coffee–chili mixture (1:1, v/v)Antiradical potential4.834.091.18
Chelating power0.220.161.43
OH• scavenging assay1.361.271.07
Reducing power2.703.730.72

Measured activity (expressed as EC50 [mg mL−1]).

Theoretical calculated activity (expressed as EC50 [mg mL−1]).

Conclusion

In this paper, a rapid and specific UPLC–MS analytical method has been developed for the determination of eight phenolic compounds from the ground chili pepper. Besides that, the presented preliminary study clearly showed that both roasted coffee and ground chili pepper are sources of compounds with multidirectional antioxidant properties. Additionally, phytochemicals from used spice possessed thermostability which justifies its potential use as an ingredient in coffee beverages. Proposed mixtures may be used in the prophylaxis or treatment of some civilization diseases linked with oxidative stress. Most importantly, conditions in the simulated gastrointestinal tract caused an increase in the activity of the individual extracts, although in most cases, synergistic interactions of aqueous extracts have changed in the antagonistic due to the digestive process. However, this astonishing issue requires further extensive studies including both in vitro and in vivo assays.

Acknowledgments

This study was funded by the National Science Center in Poland (grant number 2013/11/N/NZ9/04601).

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  • 1.

    Bors W. ; Heller W.; Michel C.; Stettmaier K. Handbook of Antioxidants (E. Cadenas and L. Packer, eds.) Marcel Dekker Inc., New York, 1996 , pp. 409 466.

    • Search Google Scholar
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    Nawrot P. ; Jordan S.; Eastwood J.; Rotstein J.; Hugenholtz A.; Feeley M. Food Addit. Cont. 2003 , 20 , 1 30.

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    Dorea J. G. ; daCosta T. H. Br. J. Nutr. 2005 , 93 , 773 782.

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