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Investigation of the interaction between soluble antioxidants in green tea and insoluble dietary fiber bound antioxidants
FRIN-05094; No of Pages 5 Food Research International xxx (2014) xxx–xxx
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Food Research International journal homepage: www.elsevier.com/locate/foodres
Investigation of the interaction between soluble antioxidants in green tea and insoluble dietary fiber bound antioxidants Ecem Evrim Çelik, Vural Gökmen ⁎ Food Engineering Department, Hacettepe University, 06800 Beytepe, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 11 January 2014 Received in revised form 18 February 2014 Accepted 22 February 2014 Available online xxxx Keywords: Green tea catechins Bound antioxidants Insoluble wheat bran Interaction of antioxidants
a b s t r a c t This work investigates the possibility of interaction between insoluble dietary fiber bound antioxidants, specifically of wheat bran, and soluble antioxidants like those provide by aqueous infusions of green tea. Solutions of pure catechins were also assayed for comparison with those naturally found in tea. To accomplish this, the aqueous and alcohol soluble fractions as well as the lipid components of wheat bran were firstly removed and the freeze-dried insoluble residue was then treated with different concentrations of green tea infusions or aqueous solutions of epicatechin (EC) and epigallocatechin-3-gallate (EGCG) for certain time. Treatment with EC (0–200 μM) had no significant effect on the antioxidant capacity of insoluble bran fraction. However, treatment with EGCG significantly (p b 0.05) increased linearly the antioxidant capacity as a function of concentration (0–100 μM). Treatment with great tea infusions (1–3 g/100 ml) also increased the resulting antioxidant capacity of insoluble bran fraction, but the effect was lesser at higher infusion concentrations. Liquid chromatography couple to mass spectrometry (LC–MS) analyses of aqueous phases after treatment indicated comparable levels of decrease in the concentrations of catechins confirming their reaction with the radical forms of antioxidants bound to insoluble bran matrix. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Tea (Camellia sinensis L.) is the second most consumed beverage worldwide after water (Yang, Wang, Lu, & Picinich, 2009). Three different types of tea (green, oolong and black) differ in terms of their production techniques, leading to the presence of different compounds. Green tea (minimally processed) is characterized by its high content of flavan3-ols, namely catechins, whereas oolong tea (partially fermented) and black tea (completely fermented) by the presence of theaflavins and thearubigins (Rein et al., 2012). Several epidemiological studies have suggested an inverse relationship between heart disease, diabetes, neurodegenerative disease and even cancer with especially green tea consumption among other tea varieties (Grove & Lambert, 2010; Higdon & Frei, 2003; Siddiqui, Afaq, Adhami, Ahmad, & Mukhtar, 2004; Williamson, Dionisi, & Renouf, 2011). The proposed beneficial effects of green tea have been attributed to its catechin content (Anesini, Ferraro, & Filip, 2008). The major catechins found in green tea are (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin-3-gallate (ECG) and (−)-epigallocatechin-3gallate (EGCG). Among them, EGCG is also the most abundant catechin present in green tea (Bhardwaj & Khanna, 2013; Lambert & Elias, 2010). Green tea catechins have proved to act as antioxidants by scavenging free radicals, inducing antioxidant enzymes or inhibiting pro-oxidant ⁎ Corresponding author. Tel.: +90 312 2977108. E-mail address: [email protected] (V. Gökmen).
enzymes (Hou et al., 2005). Once a cup of green tea is consumed, the polyphenolic antioxidants are absorbed and enter the systemic circulation rapidly which causes a significant increase in plasma antioxidant capacity (Rein et al., 2012; Skrzydlewska, Ostrowska, Farbiszewski, & Michalak, 2002). But before absorption into the plasma, free or bound green tea catechins may show activity in the extracellular medium. Immediately after ingestion, green tea catechins may start to interact with dietary fiber bound antioxidant moieties like ferulic acid in any part of the body from mouth to colon. Ferulic acid, the major polyphenol found in cereals; especially in wheat bran, is placed in the cell walls of grains esterified with arabinoxylan units (Manach, Williamson, Morand, Scalbert, & Remesy, 2005). It may undergo a dimerization reaction itself in situ and may also be found as its dimer form, diferulic acid, which fortifies the insoluble cell wall structure (Bunzel, Ralph, Marita, Hatfeld, & Steinhart, 2001; Wakabayashi, 2007). This kind of interaction may also take place between fiber bound ferulic acid and free soluble antioxidants in fluid phase. It was evidenced in our previous study that antioxidants bound to insoluble fiber matrix could be effectively regenerated in the presence of other hydrogen donating substances in liquid phase (Çelik, Gökmen, & Fogliano, 2013). This regeneration process might occur either through dimer formation or hydrogen atom transfer from soluble antioxidants to bound polyphenolic radicals. This study aimed to investigate the interaction between soluble antioxidants in green tea and insoluble dietary fiber bound antioxidants. Different concentrations of green tea catechins (infusions and pure
http://dx.doi.org/10.1016/j.foodres.2014.02.026 0963-9969/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article as: Çelik, E.E., & Gökmen, V., Investigation of the interaction between soluble antioxidants in green tea and insoluble dietary fiber bound antioxidants, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.02.026
2
E.E. Çelik, V. Gökmen / Food Research International xxx (2014) xxx–xxx
aqueous solutions) were treated with insoluble wheat bran fraction at ambient conditions. After treatment, changes in the total antioxidant capacity of insoluble fraction were determined by the QUENCHER procedure using ABTS radical probe. Changes in the concentrations of catechins in the aqueous phase were also determined by using LC–MS to confirm their interaction with polyphenolic radicals bound to insoluble bran fraction. 2. Materials and methods 2.1. Chemicals All chemicals and solvents used were of analytical grade, unless otherwise stated. Potassium peroxydisulfate, 2,2′-azinobis (3ethylbenzothiazoline-6-sulfonic acid) (ABTS), 6-hydroxy-2,5,7,8 tetramethylchroman-2 carboxylic acid (Trolox), (−) epicatechin and (−)-epigallocatechin gallate were purchased from Sigma-Aldrich Chemie (Steinheim, Germany). Ethyl alcohol, methanol, acetonitrile, and n-hexane were extra pure and purchased from Merck (Darmstadt, Germany). 2.2. Preparation of the insoluble wheat bran fraction The dietary fiber rich food matrix used in this study, wheat bran, was purchased from a local market. Ground wheat bran was washed according to the procedure described by Çelik et al. (2013) in order to remove water and alcohol soluble substances as well as lipid phase and lipid soluble substances. Washed wheat bran was freeze-dried, ground with a ceramic mortar to obtain fine powder and passed through a sieve (Endecotts Test Sieve, London, UK) of 40 (425 μm) mesh size. The final insoluble powder was tested and found to be free of soluble antioxidant compounds. It was kept frozen at −18 °C prior to experiments. 2.3. Preparation of green tea infusions and aqueous solution of pure antioxidants Green tea, purchased from a local market, was prepared in three different concentrations. Different amounts of green tea (1, 3, 5 g) were brewed with 100 mL of boiling water for 15 min. The brews were filtered through a 0.45 μm filter prior to treatment with insoluble wheat bran fraction. Stock solutions of EC and EGCG were prepared at a concentration of 0.1 M by dissolving appropriate amounts of pure compounds in distilled water. Working solutions were prepared by diluting the stock solution with distilled water to concentrations of 10, 50, 100 and 200 μM for EC, and concentrations of 5, 10, 50 and 100 μM for EGCG. 2.4. Analysis of the antioxidant capacity of insoluble wheat bran fraction After treatment with green tea infusions or solutions of pure EC and EGCG, changes in the antioxidant capacity of insoluble wheat bran fraction was measured by direct QUENCHER procedure using ABTS radical probe. The ABTS•+ radical solution was prepared according to Serpen, Gökmen, and Fogliano (2009). A portion of insoluble wheat bran (50 mg) was weighed into a test tube and the reaction was initiated by adding 10 mL of green tea infusion, or aqueous solutions of EC or EGCG prepared at different concentrations as mentioned above. The tube was vigorously shaken in an orbital shaker at a speed of 350 rpm at room temperature in dark. Aqueous solutions or green tea infusions without insoluble wheat bran were also kept under the same condition as control. After 30 min of reaction, the tube was centrifuged at 6080 g for 2 min. The supernatant was filtered trough a 0.45 μm filter and analyzed by LC–MS to monitor the changes in the concentrations of catechins. The precipitate was washed out three times with 10 ml of water to remove the remaining traces of catechins. For this purpose, the precipitate was mixed with 10 mL of water. After washing cycles,
final precipitate that was free of soluble catechins was lyophilized. 10 mg of this powder was mixed with 10 mL of ABTS•+ working solution in a test tube. The mixture was rigorously shaken for 27 min at 350 rpm in the dark using the orbital shaker. Then it was centrifuged at 6080 g for 2 min. The optically clear supernatant was transferred into a cuvette and absorbance was measured at 734 nm using a Shimadzu model 2100 variable-wavelength UV–visible spectrophotometer (Shimadzu Corp., Kyoto, Japan). The total antioxidant capacity of insoluble wheat bran was calculated by means of a calibration curve built for trolox in the concentration range between 0 and 600 μg mL−1. The results were expressed as mmol trolox equivalent antioxidant capacity per kg insoluble dietary fiber. 2.5. Analysis of the antioxidant compounds by LC–MS Chromatographic analyses were performed on an Agilent 1200 HPLC system (Waldbronn, Germany) consisting of a diode array detector, a quaternary pump, an autosampler, and a temperature controlled column oven coupled to an Agilent 6130 Quadrupole MS detector equipped with ESI source. A HICHROM 5 C18 column (250 mm × 4.6 mm, 5 μm) (Hichrom Ltd., Reading, USA) was used for the separation of phenolic compounds using a linear gradient elution program with a mobile phase containing 70% solvent A (1.0% formic acid in H2O) and 30% solvent B (1.0% formic acid in CH3CN) at a flow rate of 0.7 mL min−1 at 30 °C. Data acquisition was performed in SIM mode with negative ionization using the following MS parameters: drying gas (N2) flow of 13 L/min at 325 °C; nebulizer pressure of 40 psig; capillary voltage of 3,5 kV; and dwell time of 590 ms. Molecular ions ([M-H]−) having m/z 289 for EC, 305 for EGC, 441 for ECG and 457 for EGCG were selected for identification and quantification of corresponding compounds. The quantitation was based on individual external calibration curves of individual catechins. 2.6. Statistical analysis The results were reported as mean ± standard deviations (n = 3). Significant differences (p b 0.05) were evaluated by Duncan test after the analysis of variance (ANOVA), by using SPSS 17.0 statistical package. 3. Results and discussion 3.1. Changes in the antioxidant capacity of insoluble wheat bran fraction Fig. 1 shows the changes in the antioxidant capacity of insoluble wheat bran fraction after treating it with different concentrations of EC and EGCG for 30 min at room temperature. Compared to control, the treatment with aqueous EC solution (10–200 μM) did not cause any statistically significant change (p N 0.05) in the total antioxidant of insoluble wheat bran (Fig. 1a). Similar results were obtained when the treatment time was increased from 30 min to 60 min. However, there was a statistically significant increase (p b 005) in the antioxidant capacity of insoluble wheat bran after its treatment with aqueous solution of EGCG at different concentrations. Within 30 min of treatment, the antioxidant capacity increased linearly from 9.56 mmol trolox equivalent to 25.76 mmol trolox equivalent per kg of insoluble wheat bran when the aqueous concentration of EGCG was gradually increased from 0 to100 μM (Fig. 1b). Observed increases in the antioxidant capacity of insoluble wheat bran samples after the treatment with aqueous solutions of catechins indicate that certain bound antioxidants are naturally present in their radical forms in the microstructure of wheat bran. The regeneration of these radicals with catechins to the non-radical or reduced form seems to be favorable in aqueous medium at ambient conditions. At this point, the differences observed in the regeneration abilities of EC and EGCG might arise from the differences in their efficiencies as radical scavengers. According to the relative hierarchy, EGCG is regarded to be
Please cite this article as: Çelik, E.E., & Gökmen, V., Investigation of the interaction between soluble antioxidants in green tea and insoluble dietary fiber bound antioxidants, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.02.026
Antioxidant capacity, mmol tolox eq./kg
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30 25 20 15 10 5 0
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Fig. 2. Changes in the antioxidant capacity of insoluble wheat bran fraction after 30 min of treatment with green tea infusions at different concentrations.
of these solutions with insoluble wheat bran caused a significant decrease in the concentrations of EC and EGCG as shown in Fig. 3. The consumption rates of EC during the treatment did not follow a regular trend depending on its initial concentration (Fig. 3a). Whereas, the concentration of EGCG exponentially decreased during the treatment as its initial concentration increased (Fig. 3b). This trend was comparable with the measured increases in the antioxidant capacity of insoluble wheat bran samples during the treatment.
25 20 15 10
a 5 0 0 (control)
5 µM
10 µM
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Fig. 1. Changes in the antioxidant capacity of insoluble wheat bran fraction after 30 min of treatment with catechins at different concentrations. (a) EC, (b) EGCG.
more effective than EC which can constitute an explanation for our results (Braicu, Ladomery, Chedea, Irimie, & Berindan-Neagoe, 2013). Besides this, the mobility and the flexibility of the galloyl group of EGCG will help to improve its reaction ability with the bound antioxidant radicals by allowing it to take on variable conformations itself (Kuzuhara, Suganuma, & Fujiki, 2008). Replacing aqueous solution of catechins with green tea infusion caused remarkable changes in the antioxidant capacity of insoluble wheat bran. Compared to control, the antioxidant capacity of insoluble wheat bran was significantly higher (p b 0.05) for all infusion concentrations tested (1, 3, 5 g) as shown in Fig. 2. The highest level of antioxidant capacity (24.76 mmol trolox equivalent per kg) was attained when wheat bran was treated with the infusion prepared by brewing 1 g green tea with 100 ml of boiling water. However, there was a reverse linear correlation between the concentration of infusion and increase in the antioxidant capacity of insoluble wheat bran after 30 min of treatment. These results suggest that green tea catechins at higher concentrations loose their antioxidant efficacy during the interaction with fiber bound radicals. As previously reported, green tea catechins may behave as both antioxidants and prooxidants depending on their concentrations and free radical source (Cao, Sofic, & Prior, 1997). Accordingly, many compounds have proved to behave like prooxidants under or above a critical concentration (Fukumoto & Mazza, 2000; Maurya & Devasagayam, 2010). 3.2. Changes in the concentrations of catechins The aqueous solutions of EC and EGCG without bran material (control) were rather stable under the test conditions. However, treatment
Residual concentration, %
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Antioxidant capacity, mmol trolox eq/kg
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60 40 20 0
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Initial EGCG concentration Fig. 3. Residual concentrations of pure catechins in the aqueous phase obtained after 30 min of treatment with insoluble wheat bran fraction. (a) EC, (b) EGCG.
Please cite this article as: Çelik, E.E., & Gökmen, V., Investigation of the interaction between soluble antioxidants in green tea and insoluble dietary fiber bound antioxidants, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.02.026
E.E. Çelik, V. Gökmen / Food Research International xxx (2014) xxx–xxx
Residual concentration, %
4
120
4. Conclusion
100
It is well known that ferulic acid units are found esterified with arabinoxylan units in wheat bran. The results indicated that some of these units might be naturally in their radical forms. However, green tea catechins are very effective antioxidant agents on their regenerative modification. The fact that the different efficiency of the soluble antioxidants assayed in the present work against the insoluble bound antioxidants of wheat bran might be due to the different redox reducing potentials of each soluble antioxidant with respect to that of the reacting insoluble dietary fiber. According to the mechanism mentioned above, when hydrogen donating substances like free catechins are available in the medium, they will come into contact with bound radicals and rapidly regenerate them by either giving one of their electrons/hydrogen atoms, becoming radical themselves or constituting a covalent bond with them. In this type of interaction, the resulting insoluble fiber material would have an increased total antioxidant capacity as revealed by the results of present study. It is very important to taken this into account for the antioxidant effect of the insoluble dietary fibers when interacting with soluble antioxidants of meals into the gut and colon, where insoluble fibers like those of wheat bran can remain up to 24 h within them. This information can easily be adapted to develop new functional dietary fiber products with improved bioavailability. In addition, functional dietary programs based on the interaction mechanism of soluble and bound antioxidants can be designed for special needs.
80
60
40
20
0
EC
EGC
ECG
EGCG
Fig. 4. Residual concentrations of EC, EGC, ECG and EGCG in green tea infusion (1 g/100 mL) after 30 min of treatment with insoluble wheat bran fraction.
Fig. 4 shows the changes in the concentrations of EC, EGC, ECG and EGCG in green tea infusion (1 g/100 mL) after 30 min of treatment with insoluble wheat fraction. The consumed amounts of these compounds during the treatment varied much according to the type of catechins. Among the catechins examined, the consumption rates were in the following order: ECG N EGCG N EGC N EC. As degradation of pure catechins on their own was limited (less than 10%), their consumption during the treatment is believed to be completely dependent on their interaction with the radical forms of bound antioxidants available on the surface of insoluble matrix. The increase in the antioxidant capacity of insoluble wheat bran and the decrease in the concentrations of catechins were coherent with each other. This was a clear indication of the interaction between the soluble antioxidants in the liquid phase with the antioxidants bound to the insoluble matrix. A proposed mechanism for this kind of an interaction is shown in Fig. 5. According to this mechanism, soluble antioxidants available in a liquid phase (i.e. green tea infusion) react with the radical forms of antioxidants bound to the insoluble fiber (i.e. ferulic acid radical in wheat bran).
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Fig. 5. Proposed mechanism for the interaction between soluble antioxidants in green tea and insoluble dietary fiber bound antioxidants.
Please cite this article as: Çelik, E.E., & Gökmen, V., Investigation of the interaction between soluble antioxidants in green tea and insoluble dietary fiber bound antioxidants, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.02.026
E.E. Çelik, V. Gökmen / Food Research International xxx (2014) xxx–xxx Fukumoto, L. R., & Mazza, G. (2000). Assessing antioxidant and prooxidant activities of phenolic compounds. Journal of Agricultural and Food Chemistry, 48, 3597–3604. Grove, K. A., & Lambert, J. D. (2010). Laboratory, epidemiological, and human intervention studies show that tea (Camellia sinensis) may be useful in the prevention of obesity. Journal of Nutrition, 140, 446–453. Higdon, J. V., & Frei, V. (2003). Tea catechins and polyphenols: Health effects, metabolism and antioxidant functions. Critical Reviews in Food Science and Nutrition, 43, 89–143. Hou, Z., Sang, Y., You, H., Lee, M. J., Hong, J., Chin, K. V., et al. (2005). Mechanism of action of (−)-epigallocatechin-3-gallate: Auto-oxidation-dependent inactivation of epidermal growth factor receptor and direct effects on growth inhibition in human esophageal cancer KYSE 150 cells. Cancer Research, 65, 8049–8056. Kuzuhara, T., Suganuma, M., & Fujiki, H. (2008). Green tea catechin as a chemical chaperone in cancer prevention. Cancer Letters, 261, 18–20. Lambert, D. J., & Elias, R. J. (2010). The antioxidant and pro-oxidant activities of green tea polyphenols: A role in cancer prevention. Archives of Biochemistry and Biophysics, 501, 65–72. Manach, C., Williamson, G., Morand, C., Scalbert, A., & Remesy, C. (2005). Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. American Journal of Clinical Nutrition, 81, 230S–242S.
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Maurya, D. K., & Devasagayam, T. P. A. (2010). Antioxidant and prooxidant nature of hydroxycinnamic acid derivatives ferulic and caffeic acids. Food and Chemical Toxicology, 48, 3369–3373. Rein, J. M., Renouf, M., Cruz-Hernandez, C., Actis-Goretta, L., Thakkar, S. K., & Pinto, S. M. (2012). Bioavaliability of bioactive food compounds: A challenging journey to bioefficacy. British Journal of Clinical Pharmacology, 75, 588–602. Serpen, A., Gökmen, V., & Fogliano, V. (2009). Total antioxidant capacities of raw and cooked meats. Meat Science, 90, 60–65. Siddiqui, I. A., Afaq, F., Adhami, V. M., Ahmad, N., & Mukhtar, H. (2004). Antioxidants of the beverage tea in promotion of human health. Antioxidants and Redox Signaling, 6, 571–582. Skrzydlewska, E., Ostrowska, J., Farbiszewski, R., & Michalak, K. (2002). Protective effect of green tea against lipid peroxidation in the rat liver, blood serum and the brain. Phytomedicine, 9, 232–238. Wakabayashi, K. (2007). Regulation by gravity of ferulate formation in cell walls of rice seedlings (ferulate). JAXA Special Publication, 05–037, 2–7. Williamson, G., Dionisi, F., & Renouf, M. (2011). Flavanols from green tea and phenolic acids from coffee: Critical quantitative evaluation of the pharmacokinetic data in humans after consumption of single doses of beverages. Molecular Nutrition & Food Research, 55, 864–873. Yang, C. S., Wang, X., Lu, G., & Picinich, S. C. (2009). Cancer prevention by tea: Animal studies, molecular mechanisms and human relevance. Nature Reviews Cancer, 9, 429–439.
Please cite this article as: Çelik, E.E., & Gökmen, V., Investigation of the interaction between soluble antioxidants in green tea and insoluble dietary fiber bound antioxidants, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.02.026
Contents lists available at ScienceDirect
Food Research International journal homepage: www.elsevier.com/locate/foodres
Investigation of the interaction between soluble antioxidants in green tea and insoluble dietary fiber bound antioxidants Ecem Evrim Çelik, Vural Gökmen ⁎ Food Engineering Department, Hacettepe University, 06800 Beytepe, Ankara, Turkey
a r t i c l e
i n f o
Article history: Received 11 January 2014 Received in revised form 18 February 2014 Accepted 22 February 2014 Available online xxxx Keywords: Green tea catechins Bound antioxidants Insoluble wheat bran Interaction of antioxidants
a b s t r a c t This work investigates the possibility of interaction between insoluble dietary fiber bound antioxidants, specifically of wheat bran, and soluble antioxidants like those provide by aqueous infusions of green tea. Solutions of pure catechins were also assayed for comparison with those naturally found in tea. To accomplish this, the aqueous and alcohol soluble fractions as well as the lipid components of wheat bran were firstly removed and the freeze-dried insoluble residue was then treated with different concentrations of green tea infusions or aqueous solutions of epicatechin (EC) and epigallocatechin-3-gallate (EGCG) for certain time. Treatment with EC (0–200 μM) had no significant effect on the antioxidant capacity of insoluble bran fraction. However, treatment with EGCG significantly (p b 0.05) increased linearly the antioxidant capacity as a function of concentration (0–100 μM). Treatment with great tea infusions (1–3 g/100 ml) also increased the resulting antioxidant capacity of insoluble bran fraction, but the effect was lesser at higher infusion concentrations. Liquid chromatography couple to mass spectrometry (LC–MS) analyses of aqueous phases after treatment indicated comparable levels of decrease in the concentrations of catechins confirming their reaction with the radical forms of antioxidants bound to insoluble bran matrix. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Tea (Camellia sinensis L.) is the second most consumed beverage worldwide after water (Yang, Wang, Lu, & Picinich, 2009). Three different types of tea (green, oolong and black) differ in terms of their production techniques, leading to the presence of different compounds. Green tea (minimally processed) is characterized by its high content of flavan3-ols, namely catechins, whereas oolong tea (partially fermented) and black tea (completely fermented) by the presence of theaflavins and thearubigins (Rein et al., 2012). Several epidemiological studies have suggested an inverse relationship between heart disease, diabetes, neurodegenerative disease and even cancer with especially green tea consumption among other tea varieties (Grove & Lambert, 2010; Higdon & Frei, 2003; Siddiqui, Afaq, Adhami, Ahmad, & Mukhtar, 2004; Williamson, Dionisi, & Renouf, 2011). The proposed beneficial effects of green tea have been attributed to its catechin content (Anesini, Ferraro, & Filip, 2008). The major catechins found in green tea are (−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin-3-gallate (ECG) and (−)-epigallocatechin-3gallate (EGCG). Among them, EGCG is also the most abundant catechin present in green tea (Bhardwaj & Khanna, 2013; Lambert & Elias, 2010). Green tea catechins have proved to act as antioxidants by scavenging free radicals, inducing antioxidant enzymes or inhibiting pro-oxidant ⁎ Corresponding author. Tel.: +90 312 2977108. E-mail address: [email protected] (V. Gökmen).
enzymes (Hou et al., 2005). Once a cup of green tea is consumed, the polyphenolic antioxidants are absorbed and enter the systemic circulation rapidly which causes a significant increase in plasma antioxidant capacity (Rein et al., 2012; Skrzydlewska, Ostrowska, Farbiszewski, & Michalak, 2002). But before absorption into the plasma, free or bound green tea catechins may show activity in the extracellular medium. Immediately after ingestion, green tea catechins may start to interact with dietary fiber bound antioxidant moieties like ferulic acid in any part of the body from mouth to colon. Ferulic acid, the major polyphenol found in cereals; especially in wheat bran, is placed in the cell walls of grains esterified with arabinoxylan units (Manach, Williamson, Morand, Scalbert, & Remesy, 2005). It may undergo a dimerization reaction itself in situ and may also be found as its dimer form, diferulic acid, which fortifies the insoluble cell wall structure (Bunzel, Ralph, Marita, Hatfeld, & Steinhart, 2001; Wakabayashi, 2007). This kind of interaction may also take place between fiber bound ferulic acid and free soluble antioxidants in fluid phase. It was evidenced in our previous study that antioxidants bound to insoluble fiber matrix could be effectively regenerated in the presence of other hydrogen donating substances in liquid phase (Çelik, Gökmen, & Fogliano, 2013). This regeneration process might occur either through dimer formation or hydrogen atom transfer from soluble antioxidants to bound polyphenolic radicals. This study aimed to investigate the interaction between soluble antioxidants in green tea and insoluble dietary fiber bound antioxidants. Different concentrations of green tea catechins (infusions and pure
http://dx.doi.org/10.1016/j.foodres.2014.02.026 0963-9969/© 2014 Elsevier Ltd. All rights reserved.
Please cite this article as: Çelik, E.E., & Gökmen, V., Investigation of the interaction between soluble antioxidants in green tea and insoluble dietary fiber bound antioxidants, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.02.026
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E.E. Çelik, V. Gökmen / Food Research International xxx (2014) xxx–xxx
aqueous solutions) were treated with insoluble wheat bran fraction at ambient conditions. After treatment, changes in the total antioxidant capacity of insoluble fraction were determined by the QUENCHER procedure using ABTS radical probe. Changes in the concentrations of catechins in the aqueous phase were also determined by using LC–MS to confirm their interaction with polyphenolic radicals bound to insoluble bran fraction. 2. Materials and methods 2.1. Chemicals All chemicals and solvents used were of analytical grade, unless otherwise stated. Potassium peroxydisulfate, 2,2′-azinobis (3ethylbenzothiazoline-6-sulfonic acid) (ABTS), 6-hydroxy-2,5,7,8 tetramethylchroman-2 carboxylic acid (Trolox), (−) epicatechin and (−)-epigallocatechin gallate were purchased from Sigma-Aldrich Chemie (Steinheim, Germany). Ethyl alcohol, methanol, acetonitrile, and n-hexane were extra pure and purchased from Merck (Darmstadt, Germany). 2.2. Preparation of the insoluble wheat bran fraction The dietary fiber rich food matrix used in this study, wheat bran, was purchased from a local market. Ground wheat bran was washed according to the procedure described by Çelik et al. (2013) in order to remove water and alcohol soluble substances as well as lipid phase and lipid soluble substances. Washed wheat bran was freeze-dried, ground with a ceramic mortar to obtain fine powder and passed through a sieve (Endecotts Test Sieve, London, UK) of 40 (425 μm) mesh size. The final insoluble powder was tested and found to be free of soluble antioxidant compounds. It was kept frozen at −18 °C prior to experiments. 2.3. Preparation of green tea infusions and aqueous solution of pure antioxidants Green tea, purchased from a local market, was prepared in three different concentrations. Different amounts of green tea (1, 3, 5 g) were brewed with 100 mL of boiling water for 15 min. The brews were filtered through a 0.45 μm filter prior to treatment with insoluble wheat bran fraction. Stock solutions of EC and EGCG were prepared at a concentration of 0.1 M by dissolving appropriate amounts of pure compounds in distilled water. Working solutions were prepared by diluting the stock solution with distilled water to concentrations of 10, 50, 100 and 200 μM for EC, and concentrations of 5, 10, 50 and 100 μM for EGCG. 2.4. Analysis of the antioxidant capacity of insoluble wheat bran fraction After treatment with green tea infusions or solutions of pure EC and EGCG, changes in the antioxidant capacity of insoluble wheat bran fraction was measured by direct QUENCHER procedure using ABTS radical probe. The ABTS•+ radical solution was prepared according to Serpen, Gökmen, and Fogliano (2009). A portion of insoluble wheat bran (50 mg) was weighed into a test tube and the reaction was initiated by adding 10 mL of green tea infusion, or aqueous solutions of EC or EGCG prepared at different concentrations as mentioned above. The tube was vigorously shaken in an orbital shaker at a speed of 350 rpm at room temperature in dark. Aqueous solutions or green tea infusions without insoluble wheat bran were also kept under the same condition as control. After 30 min of reaction, the tube was centrifuged at 6080 g for 2 min. The supernatant was filtered trough a 0.45 μm filter and analyzed by LC–MS to monitor the changes in the concentrations of catechins. The precipitate was washed out three times with 10 ml of water to remove the remaining traces of catechins. For this purpose, the precipitate was mixed with 10 mL of water. After washing cycles,
final precipitate that was free of soluble catechins was lyophilized. 10 mg of this powder was mixed with 10 mL of ABTS•+ working solution in a test tube. The mixture was rigorously shaken for 27 min at 350 rpm in the dark using the orbital shaker. Then it was centrifuged at 6080 g for 2 min. The optically clear supernatant was transferred into a cuvette and absorbance was measured at 734 nm using a Shimadzu model 2100 variable-wavelength UV–visible spectrophotometer (Shimadzu Corp., Kyoto, Japan). The total antioxidant capacity of insoluble wheat bran was calculated by means of a calibration curve built for trolox in the concentration range between 0 and 600 μg mL−1. The results were expressed as mmol trolox equivalent antioxidant capacity per kg insoluble dietary fiber. 2.5. Analysis of the antioxidant compounds by LC–MS Chromatographic analyses were performed on an Agilent 1200 HPLC system (Waldbronn, Germany) consisting of a diode array detector, a quaternary pump, an autosampler, and a temperature controlled column oven coupled to an Agilent 6130 Quadrupole MS detector equipped with ESI source. A HICHROM 5 C18 column (250 mm × 4.6 mm, 5 μm) (Hichrom Ltd., Reading, USA) was used for the separation of phenolic compounds using a linear gradient elution program with a mobile phase containing 70% solvent A (1.0% formic acid in H2O) and 30% solvent B (1.0% formic acid in CH3CN) at a flow rate of 0.7 mL min−1 at 30 °C. Data acquisition was performed in SIM mode with negative ionization using the following MS parameters: drying gas (N2) flow of 13 L/min at 325 °C; nebulizer pressure of 40 psig; capillary voltage of 3,5 kV; and dwell time of 590 ms. Molecular ions ([M-H]−) having m/z 289 for EC, 305 for EGC, 441 for ECG and 457 for EGCG were selected for identification and quantification of corresponding compounds. The quantitation was based on individual external calibration curves of individual catechins. 2.6. Statistical analysis The results were reported as mean ± standard deviations (n = 3). Significant differences (p b 0.05) were evaluated by Duncan test after the analysis of variance (ANOVA), by using SPSS 17.0 statistical package. 3. Results and discussion 3.1. Changes in the antioxidant capacity of insoluble wheat bran fraction Fig. 1 shows the changes in the antioxidant capacity of insoluble wheat bran fraction after treating it with different concentrations of EC and EGCG for 30 min at room temperature. Compared to control, the treatment with aqueous EC solution (10–200 μM) did not cause any statistically significant change (p N 0.05) in the total antioxidant of insoluble wheat bran (Fig. 1a). Similar results were obtained when the treatment time was increased from 30 min to 60 min. However, there was a statistically significant increase (p b 005) in the antioxidant capacity of insoluble wheat bran after its treatment with aqueous solution of EGCG at different concentrations. Within 30 min of treatment, the antioxidant capacity increased linearly from 9.56 mmol trolox equivalent to 25.76 mmol trolox equivalent per kg of insoluble wheat bran when the aqueous concentration of EGCG was gradually increased from 0 to100 μM (Fig. 1b). Observed increases in the antioxidant capacity of insoluble wheat bran samples after the treatment with aqueous solutions of catechins indicate that certain bound antioxidants are naturally present in their radical forms in the microstructure of wheat bran. The regeneration of these radicals with catechins to the non-radical or reduced form seems to be favorable in aqueous medium at ambient conditions. At this point, the differences observed in the regeneration abilities of EC and EGCG might arise from the differences in their efficiencies as radical scavengers. According to the relative hierarchy, EGCG is regarded to be
Please cite this article as: Çelik, E.E., & Gökmen, V., Investigation of the interaction between soluble antioxidants in green tea and insoluble dietary fiber bound antioxidants, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.02.026
Antioxidant capacity, mmol tolox eq./kg
E.E. Çelik, V. Gökmen / Food Research International xxx (2014) xxx–xxx
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Fig. 2. Changes in the antioxidant capacity of insoluble wheat bran fraction after 30 min of treatment with green tea infusions at different concentrations.
of these solutions with insoluble wheat bran caused a significant decrease in the concentrations of EC and EGCG as shown in Fig. 3. The consumption rates of EC during the treatment did not follow a regular trend depending on its initial concentration (Fig. 3a). Whereas, the concentration of EGCG exponentially decreased during the treatment as its initial concentration increased (Fig. 3b). This trend was comparable with the measured increases in the antioxidant capacity of insoluble wheat bran samples during the treatment.
25 20 15 10
a 5 0 0 (control)
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Fig. 1. Changes in the antioxidant capacity of insoluble wheat bran fraction after 30 min of treatment with catechins at different concentrations. (a) EC, (b) EGCG.
more effective than EC which can constitute an explanation for our results (Braicu, Ladomery, Chedea, Irimie, & Berindan-Neagoe, 2013). Besides this, the mobility and the flexibility of the galloyl group of EGCG will help to improve its reaction ability with the bound antioxidant radicals by allowing it to take on variable conformations itself (Kuzuhara, Suganuma, & Fujiki, 2008). Replacing aqueous solution of catechins with green tea infusion caused remarkable changes in the antioxidant capacity of insoluble wheat bran. Compared to control, the antioxidant capacity of insoluble wheat bran was significantly higher (p b 0.05) for all infusion concentrations tested (1, 3, 5 g) as shown in Fig. 2. The highest level of antioxidant capacity (24.76 mmol trolox equivalent per kg) was attained when wheat bran was treated with the infusion prepared by brewing 1 g green tea with 100 ml of boiling water. However, there was a reverse linear correlation between the concentration of infusion and increase in the antioxidant capacity of insoluble wheat bran after 30 min of treatment. These results suggest that green tea catechins at higher concentrations loose their antioxidant efficacy during the interaction with fiber bound radicals. As previously reported, green tea catechins may behave as both antioxidants and prooxidants depending on their concentrations and free radical source (Cao, Sofic, & Prior, 1997). Accordingly, many compounds have proved to behave like prooxidants under or above a critical concentration (Fukumoto & Mazza, 2000; Maurya & Devasagayam, 2010). 3.2. Changes in the concentrations of catechins The aqueous solutions of EC and EGCG without bran material (control) were rather stable under the test conditions. However, treatment
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Initial EGCG concentration Fig. 3. Residual concentrations of pure catechins in the aqueous phase obtained after 30 min of treatment with insoluble wheat bran fraction. (a) EC, (b) EGCG.
Please cite this article as: Çelik, E.E., & Gökmen, V., Investigation of the interaction between soluble antioxidants in green tea and insoluble dietary fiber bound antioxidants, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.02.026
E.E. Çelik, V. Gökmen / Food Research International xxx (2014) xxx–xxx
Residual concentration, %
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4. Conclusion
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It is well known that ferulic acid units are found esterified with arabinoxylan units in wheat bran. The results indicated that some of these units might be naturally in their radical forms. However, green tea catechins are very effective antioxidant agents on their regenerative modification. The fact that the different efficiency of the soluble antioxidants assayed in the present work against the insoluble bound antioxidants of wheat bran might be due to the different redox reducing potentials of each soluble antioxidant with respect to that of the reacting insoluble dietary fiber. According to the mechanism mentioned above, when hydrogen donating substances like free catechins are available in the medium, they will come into contact with bound radicals and rapidly regenerate them by either giving one of their electrons/hydrogen atoms, becoming radical themselves or constituting a covalent bond with them. In this type of interaction, the resulting insoluble fiber material would have an increased total antioxidant capacity as revealed by the results of present study. It is very important to taken this into account for the antioxidant effect of the insoluble dietary fibers when interacting with soluble antioxidants of meals into the gut and colon, where insoluble fibers like those of wheat bran can remain up to 24 h within them. This information can easily be adapted to develop new functional dietary fiber products with improved bioavailability. In addition, functional dietary programs based on the interaction mechanism of soluble and bound antioxidants can be designed for special needs.
80
60
40
20
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EC
EGC
ECG
EGCG
Fig. 4. Residual concentrations of EC, EGC, ECG and EGCG in green tea infusion (1 g/100 mL) after 30 min of treatment with insoluble wheat bran fraction.
Fig. 4 shows the changes in the concentrations of EC, EGC, ECG and EGCG in green tea infusion (1 g/100 mL) after 30 min of treatment with insoluble wheat fraction. The consumed amounts of these compounds during the treatment varied much according to the type of catechins. Among the catechins examined, the consumption rates were in the following order: ECG N EGCG N EGC N EC. As degradation of pure catechins on their own was limited (less than 10%), their consumption during the treatment is believed to be completely dependent on their interaction with the radical forms of bound antioxidants available on the surface of insoluble matrix. The increase in the antioxidant capacity of insoluble wheat bran and the decrease in the concentrations of catechins were coherent with each other. This was a clear indication of the interaction between the soluble antioxidants in the liquid phase with the antioxidants bound to the insoluble matrix. A proposed mechanism for this kind of an interaction is shown in Fig. 5. According to this mechanism, soluble antioxidants available in a liquid phase (i.e. green tea infusion) react with the radical forms of antioxidants bound to the insoluble fiber (i.e. ferulic acid radical in wheat bran).
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Please cite this article as: Çelik, E.E., & Gökmen, V., Investigation of the interaction between soluble antioxidants in green tea and insoluble dietary fiber bound antioxidants, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.02.026
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Please cite this article as: Çelik, E.E., & Gökmen, V., Investigation of the interaction between soluble antioxidants in green tea and insoluble dietary fiber bound antioxidants, Food Research International (2014), http://dx.doi.org/10.1016/j.foodres.2014.02.026