Total antioxidant capacity

ANTIOXIDANTS JAVIER GARCÍA-LOMILLO 1 *, M LUISA GONZÁLEZ-SANJOSÉ 1 *Corresponding author 1. Department of Biotechnology and Food Science, Faculty of Sciences, University of Burgos, Plaza de Misael Bañuelos, 09001 Burgos, Spain. Javier Garcìa-Lomillo Total antioxidant capacity of decaffeinated beverages KEYWORDS: antioxidant activity, coffee, tea, decaffeinated beverages Decaffeinated beverages are rising in popularity among coffee and tea drinkers; however, little is known Abstract about the effect of the decaffeination process on the total antioxidant capacity (TAC). The aim of this work was to determine whether there is a significant difference between regular beverages and their decaffeinated equivalents in terms of the TAC. Colour, total phenolic content, total catechin content, and antioxidant activities (ABTS+, FRAP, DPPH) were determined in five commercial black teas and six commercial instant coffees as well as their respective decaffeinated products from the same brand. Only one of the brands showed significant differences in all the tests and no clear differences were reported. In the factorial analysis, it was observed that samples were associated according to their brands rather than according to their caffeine content. Consequently, factors associated with the brand are in fact, what determine the TAC of the beverage. INTRODUCTION Coffee and tea are two of the most consumed drinks in the United Kingdom. According to the last European coffee report, 3 kg of coffee were consumed per person in 2011; which means that more than 60 million cups of coffee are consumed per day in the UK. Because it is easier to make, instant coffee is consumed more than any other type of coffee (75% of coffee is drunk as instant coffee). As far as tea is concerned, the United Kingdom has the second highest tea consumption rate in the world (2.1 kg of tea per person). More than 165 million cups of tea are drunk per day, according to the estimations of the UK Tea Council (1). The demand for decaffeinated coffee and tea is increasing and their intake represents around 15% of the coffee and tea market. Consumers are aware of caffeine effects on health which could explain the increase in the demand for decaffeinated products. It is well known that sleep is affected by consumption of caffeine. Moreover, nervousness, agitation, and anxiety are caused by consumption of non-decaffeinated coffee and tea (2). In addition to that, caffeine has been directly associated with several types of cancer (3), coronary disease(4), and abortion (5). However, these effects are not conclusive yet and further research is required. The decaffeination process causes several changes to the physical and chemical characteristics of the final product. To avoid the loss of the flavour and aroma produced in coffee roasting, the decaffeination process is conducted on the green coffee beans. Thus, changes produced during the decaffeination process may have an influence on the following steps of coffee production: roasting, extraction and drying. They are crucial since total antioxidant capacity (TAC) and characteristics of the final product are developed during these processes. In the case of tea, decaffeination of green leaves produces an unusual taste. As a result, the decaffeination process is carried out after the tea leaves have already been through the manufacturing process. In this case, antioxidants may be removed with the caffeine, and others may be modified by heat treatment applied in the decaffeination process. Due to the complex nature of the antioxidants and the wide range of reactions in which they are involved, there is no consensus of opinions on the way the TAC should be measured. Chemical assays such as 2,2 -azino-bis(3-ethylbenzothiazoline-6 sulfonic acid) (ABTS) radical-scavenging, 2,2-diphenyl-1- picrylhydrazyl (DPPH) radical-scavenging and ferric reducing antioxidant power (FRAP) have been widely used to measure the TAC of coffee and tea beverages (6-16). Although their results do not have to be necessarily correlated with the health benefits of coffee and tea, these assays provides a reliable quantitation of the capacity to inhibit the oxidation in in vitro conditions (17). There is an increasing tendency towards the consumption of decaffeinated drinks; however, little attention has been paid to the changes on the TAC due to the decaffeination process. Although some studies have already measured the TAC of decaffeinated drinks, none of them has focused on the comparison between decaffeinated and regular beverages. The aim of this work was to determine whether there is a significant difference between regular beverages and their decaffeinated equivalents in terms of the TAC. MATERIALS AND METHODS Chemicals and equipment ABTS, potassium persulphate (K 2 O 8 S 2 ) and 6-hydroxyl-2,5,7,8- tetramethyl-2-carboxylic acid (Trolox), were purchased from Calbiochem (San Diego, U.S.A.) and Acros (New Jersey, U.S.A.), respectively. 2,4,6-Tris(2-pyridyl)-S-triazine (TPTZ) and DPPH were obtained from Sigma-Aldrich (St. Lois, USA). Sodium carbonate, sodium acetate, acetic acid and ferric (III) chloride (FeCl 3 ) were Agro FOOD Industry Hi Tech - vol 25(2) - March/April 2014 43

procured from Panreac (Barcelona, Spain). Folin-Ciocalteu reagent, hydrochloric acid (HCl) and vanillin were provided by Merck (Darmstadt, Germany). The absorbance readings were made on a Beckman DU 650 spectrophotometer. Samples Six soluble coffees, five black teas and their respective decaffeinated products from the same brand were purchased from different local supermarkets of Leeds (United Kingdom) (Table 1). Coffee samples were prepared by pouring 250 ml of boiling water on 2 grams of instant coffee. To brew tea samples, one tea bag was added to 250 ml of boiling water for 10 minutes. All samples were allowed to cool until they reached room temperature and then, analysed. Table 1. Samples studied: name and information declared by the companies. a T2 declared that the tea blend used in the decaffeinated product is different from that used for nondecaffeinated product. reduction of ABTS radical by antioxidant compounds (21). Forty μl of sample were mixed with 2 ml of ABTS reagent directly in the cuvette and then shaken manually. After 10 minutes, absorbance at 734 nm was measured and compared with a control sample. DPPH method. This method measures the decrease of DPPH radical due to its reaction with antioxidant compounds from the sample (22). DPPH reagent was prepared with an absorbance of 0.7±0.1 at 517 nm. Twenty μl of sample were added to 980 μl of DPPH reagent and incubated during 30 minutes in case of tea and 90 minutes in case of coffee. FRAP method. Ferric reducing antioxidant power (FRAP) method measures the ability of a sample to reduce a ferrictripyridyltriazine complex (23). Nine hundred seventy μl of FRAP reagent were mixed with 30 μl of sample and incubated at 37ºC for 30 minutes. The formation of the ferrous ion was quantified spectrophotometrically at 593 nm. Statistical analysis All determinations for tea samples were performed in duplicate for triplicate extractions, giving six observations for each sample. In the case of coffees, they were brewed in duplicate with two determinations for each sample, giving four observations for each sample. LSD test was used to detect significant differences between means at a confidence level of 95%. Factor analysis was conducted extracting those factors that had eigenvalues greater than or equal to 1.0. A varimax rotation was performed in order to simplify the explanation of the factors. Statistical analyses were carried out using the Statgraphic Computer System program version Centurion XVI.I. RESULTS AND DISCUSION Parameters analysed Colour measurement The brewed samples were diluted with distilled water (1:20) and then, the absorbance at 360, 420 and 440 nm was recorded using quartz cuvettes. These measurements are indicators of the colour of the sample and the absorbance at 360 nm was used as an estimation of the melanoidins content (18). Estimation of the total phenolic content (TPC) by the Folin Ciocalteu assay Folin-Ciocalteu reagent was used to determine the amount of the total phenols following the Singleton and Rossi method. (19) Results are expressed as mg gallic acid by gram of sample. Estimation of the total catechin content (TCC) by the Vanillin assay One ml of the sample was added to a test tube with 2 ml of vanillin reagent and 7 ml of HCl (26%). Absorbance at 500 nm was measured after 25 minutes of reaction time according to the method of Swain and Hillis (20). Determination of total antioxidant capacity ABTS method. This method is based on reading the decolouration (absorbance at 734 nm) after the Physiologically speaking, an antioxidant could be defined as any substance which is capable to inhibit oxidative processes (24). However, there is an antioxidant/prooxidant balance and no compound acts always as antioxidant. Since foodstuff may be complex matrices, several analyses are required to assay the TAC of samples. In this study, FRAP, DPPH and ABTS methods were chosen to assay the TAC of coffee and tea. Furthermore, colour, TPC and TCC were measured, as they are indicators of the amount of different antioxidants in the beverages that were studied (Tables 2 and 3). Table 2. Colour, total phenolic content (TPC), total catechin content (TCC), 2,2 -azino-bis(3- ethylbenzothiazoline-6 sulfonic acid) radical-scavenging activity (ABTS), 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity (DPPH) and ferric reducing antioxidant power (FRAP) for regular (Reg) and decaffeinated (Dec) tea samples listed in Table 1. Means (±SD) of duplicate measurements for three separate runs (n = 6). The letters a and b represent significant statistical differences between regular (Reg) and decaffeinated (Dec) samples from the same tea brand 44 Agro FOOD Industry Hi Tech - vol 25(2) - March/April 2014

Table 3. Colour, total phenolic content (TPC), total catechin content (TCC), 2,2 -azino-bis(3- ethylbenzothiazoline-6 sulfonic acid) radical-scavenging activity (ABTS), 2,2-diphenyl-1- picrylhydrazyl radical-scavenging activity (DPPH) and ferric reducing antioxidant power (FRAP) for regular (Reg) and decaffeinated (Dec) coffee samples listed in Table 1. Means (±SD) of duplicate measurements for two separate runs (n = 4). The letters a and b represent significant statistical differences between regular (Reg) and decaffeinated (Dec) samples from the same coffee brand Colour measurement The results from the colour tests did not show any surprising results. Coffees were darker than teas and no relevant differences were detected between decaffeinated and regular products (Tables 2 and 3). Data from the three wavelengths were similar and higher values were those from 360 nm, showing a predominant yellow tonality with low influence of red colours. Determination of total polyphenols Data from the TPC analysis showed that soluble coffees are richer in polyphenols than teas (Tables 2 and 3). The results showed that three regular coffees and three regular teas had statistically higher TPC content than their correspondent decaffeinated samples. On the other hand, one decaffeinated coffee reported higher TPC content than its regular sample. These results agree with those published by Sánchez-González et al. (8) and Chu et al. (25), which did not find any relevant difference in TPC levels of regular and decaffeinated instant coffees. However, Alves et al. (11) suggested that the decaffeination process does have an influence on TPC levels, as they detected greater amounts of TPC in regular coffee than in decaffeinated ones. In the case of the coffee samples, there were a negative correlation between TPC and colour (p value = 0.0098). This could be explained by the phenol incorporation to the structure of the melanoidins during roasting process. The stronger the roasting process, the higher the amount of polyphenols degraded, the higher the amount of melanoidins produced and the darker the melanoidins formed (26). Determination of total catechins Regarding the catechins content, all the samples showed a relevant amount of catechins measured by the Vanillin-HCL assay (Tables 2 and 3). 8 samples reported higher TCC in their non-decaffeinated samples, but in 3 beverages the opposite results was observed. The highest difference was found in T3 (around 44% less in their decaffeinated samples). Liang et al. (27) and Perva-Uzunalic et al. (28) also reported losses of 5% and 17%, respectively. The authors suggested that prior moistening and posterior drying were the main causes of these losses. There was a strong correlation (p<0.001) between TPC and TCC contents in tea as catechins are the main group of polyphenols in tea (29). However, this correlation was not significant in coffee because the major phenolic compounds in coffee are not catechins but caffeic acid, chlorogenic acid and ferulic acid (9). Determination of total antioxidant capacity Firstly, it is worth pointing out that caffeine consumes neither ABTS nor DPPH radicals nor ferric ion (7, 13) (this fact was also corroborated in this work conducting the antioxidant test with a solution of caffeine at 10 g/l and no reaction was reported). Therefore, the differences observed in the TAC of regular and decaffeinated samples were not due to the content of caffeine. ABTS Method. Regarding the results from the ABTS method; statistical differences were found in 6 samples (3 teas and 3 coffees). The regular samples of those brands reported higher ABTS values than their decaffeinated equivalents (Tables 2 and 3). On the other hand, decaffeinated T5 showed higher activity against ABTS radical than its regular equivalent. These results comply with those obtained by Parras et al. (10) and by Sanchéz-Gonzalez et al.. (8) In these studies, there were generally no differences between the scavenger activity against ABTS radical of regular coffee and that of decaffeinated coffee. DPPH method. All the samples studied, regardless of brand, were effective scavengers of DPPH radicals (Tables 2 and 3). Although differences between regular and decaffeinated samples in the DPPH value were observed in three products, no significant differences in the average were detected. The greatest difference was observed in T3 as the decaffeinated sample reported 30% less TAC than its regular equivalent. This decrease was similar to the one reported in the study of Du Toit (6) with the same tea brand. FRAP method. All 22 samples showed reductive capacity against Fe (III) (Tables 2 and 3). Although the averages of decaffeinated and regular beverages were not statistically different, 4 teas and 3 coffee products showed greater FRAP value in their regular samples than in their decaffeinated ones. Sánchez-Gonzalez et al. (8) tested the iron reducing activity of decaffeinated coffee and regular coffee brewed by three different methods and they did not find any significant differences. On the other hand, a significant decrease (5.9%-19.3%) was reported by Moreira et al. (7) but no relevant explanation was cited. TPC were strongly and directly correlated with the TAC of tea samples measured by DPPH, FRAP and ABTS tests (p-value < 0.0001). Moreover, the results from the DPPH, FRAP and ABTS tests were also statistically correlated. Jayasekera et al. (12) also found that those correlations were significant for fermented teas. Nevertheless, the correlations between TAC assays and TPC were not significant for unfermented dry season teas. These results suggest that polyphenol compounds (catechins, thearubigens and theaflavins) are a major contributor to the radical-scavenging activity and ferric reducing antioxidant power of black tea. Unlike in the tea results, no strong correlations between the TPC and TAC assays were found in the results from coffee analysis. Although several studies (8, 14) have found that the TPC contents were positively correlated with ABTS, DPPH and FRAP values, a similar conclusion than this work has been previously reported by Brezová et al.. (13) The authors suggested that melanoidins have greater importance in the TAC than polyphenols in instant coffee, as polyphenols are partly degraded during the extraction and drying processes of instant coffee. Agro FOOD Industry Hi Tech - vol 25(2) - March/April 2014 45

Multifactorial analysis A factor analysis was conducted to detect multiple associations of variables and to check natural grouping of samples. The factorial analysis of tea results gave two factors with eigenvalues higher than 1.0. Factor 2 was able to differentiate two brands (T1 and T3) as their scores were lower than the scores of the others brands (Figure 1), especially in ABTS and colour, which were the main variables for this factor. On the other hand, factor 1 was mainly correlated with TCC, DPPH, FRAP and TPC. This factor showed differences between T1 (high values) and T3 (low values). CONCLUSIONS In summary, both regular and decaffeinated drinks showed a remarkable TAC measured by ABTS, DPPH and FRAP assays. As regular coffee and tea have been widely considered as a good source of antioxidants in the diet and no difference between decaffeinated and regular beverages was detected in this work, it can be concluded that decaffeinated beverages are also an appropriate source of antioxidants. There are, however, other factors associated with the brand which determine the antioxidant profile of the final product. It is worth reminding that the methods used in this work were a measure of TAC in in vitro conditions and the effect of decaffeination on health effects of coffee and tea should also be checked. Figure 1. Factor analysis of 5 nondecaffeinated teas (filled symbols) and their decaffeinated equivalents from the same brand (open symbols). ACKNOWLEDGEMENTS The author gratefully acknowledges Gary Williamson for all the help provided during his stay at University of Leeds. The PhD grant of Javier Garcia is funded by the PIRTU programme Consejería de Educación de la Junta de Castilla y León and the European Social Fund. The factor analysis was also carried out with coffee results. Three factors reported an eigenvalue higher than 1.0. Factor 1 was able to distinguish C3 (low score) and C1 (high score) from the others samples (Figure 2). FRAP and colour reported positive effect, whereas catechins and TPC had negative effect on Factor 1. Factor 2, that was mainly correlated with colour (positively) and TCC (negatively), was able to identify as different C4. Although factor 3 had an eigenvalue higher than 1.0, it did not give any additional information. Figure 2. Factor analysis of 6 nondecaffeinated coffees (filled symbols) and their decaffeinated equivalents from the same brand (open symbols). From the results of two tea brands (T1 and T3) and two coffee brands (C1 and C4), it can be observed that decaffeinated samples were separated from regular samples. However, beverages were generally distributed according to their brand rather than according to their caffeine content. Consequently, although changes may take place during the decaffeination process, these changes are actually less significant than the factors associated to the brand. Among the factors that have been reported to affect the TAC of instant coffee, are raw material (14, 15), roasting process (7, 11, 16), solubilisation process (15) and storage (30). As far as tea is concerned, it has been reported that TAC of tea is clearly influenced by the season and the plantation in which tea is grown (12). REFERENCES AND NOTES 1. United Kingdom Tea Council: http://www.tea.co.uk/ (last checked on Jun. 8 th 2013). 2. A. Nehlig, J.-L. Daval, et al., Brain Res. Rev., 17, 139-170 (1992). 3. G. M. Al-Hachim, Eur J Obstet Gynecol Reprod Biol, 31, 237-247 (1989). 4. M. Wei, C. A. Macera, et al., J. Clin. Epidemiol., 48, 1189-1196 (1995). 5. M. A. Klebanoff, R. J. Levine, et al., New Engl. J. Med., 341, 1639-1644 (1999). 6. R. Du Toit, Y. Volsteedt, et al., Toxicology, 166, 63-69 (2001). 7. D. P. Moreira, M. C. Monteiro, et al., J. Agric. Food Chem., 53, 1399-1402 (2005). 8. I. Sánchez-González, A. Jiménez-Escrig, et al., Food Chem., 90, 133-139 (2005). 9. J. Á. Gómez-Ruiz, D. S. Leake, et al., J. Agric. Food Chem., 55, 6962-6969 (2007). 10. P. Parras, M. Martínez-Tomé, et al., Food Chem., 102, 582-592 (2007). 11. R. C. Alves, A. S. G. Costa, et al., J. Agric. Food Chem., 58, 12221-12229 (2010). 12. S. Jayasekera, A. L. Molan, et al., Food Chem., 125, 536-541 (2011). 13. V. Brezová, A. Slebodová, et al., Food Chem., 114, 859-868 (2009). 14. M. Naranjo, L. T. Vélez, et al., Rev. Cubana Plant. Med., 16, 164-173 (2011). 15. J. A. Vignoli, D. G. Bassoli, et al., Food Chem., 124, 863-868 (2011). 16. R. Del Pino-García, M.L. González-Sanjosé, et al., J. Agric. Food Chem., 60, 10530-10539 (2012) 17. D. Huang, B. Ou, et al., J. Agric. Food Chem., 53, 1841-1856 (2005). 18. M. D. Rivero-Pérez, S. Pérez-Magariño, et al., Anal. Chim. Acta, 458, 169-175 (2002). 19. V. L. Singleton and J. A. Rossi, Jr., Am. J. Enol. Vitic., 16, 144-158 (1965). 20. T. Swain and W. E. Hillis, J. Sci. Food Agric., 10, 63-68 (1959). 21. R. Re, N. Pellegrini, et al., Free Radical Biol. Med., 26, 1231-1237 (1999). 22. W. Brand-Williams, M. E. Cuvelier, et al., Food Sci. Technol., 28, 25-30 (1995). 23. I. F. F. Benzie and J. J. Strain, Anal. Biochem., 239, 70-76 (1996). 24. J. Pokorný, Eur. J. Lipid Sci. Technol., 109, 629-642 (2007). 25. Y.-F. Chu, Y. Chen, et al., Food Chem., 124, 914-920 (2011). 26. S. Pérez-Magariño, M. D. Rivero, et al., Czech Journal of Food Science 18, 108-109 (2000). 27. H. Liang, Y. Liang, et al., Food Chem., 101, 1451-1456 (2007). 28. A. Perva-Uzunalic, M. Skerget, et al., Food Chem., 96, 597-605 (2006). 29. Y. Wang and C.-T. Ho, J. Agric. Food Chem., 57, 8109-8114 (2009). 30. M. Anese and M. C. Nicoli, J. Agric. Food Chem., 51, 942-946 (2003). 46 Agro FOOD Industry Hi Tech - vol 25(2) - March/April 2014