The methods based on the evaluation of the capacity of antioxidants or their complex mixtures to reduce metals are named FRAP (ferric reducing antioxidant powder) and CUPRAC (cupric reducing antioxidant powder).
FRAP Method
FRAP assay was originally developed by Benzie et al.[67] to measure the reducing power of plasma. However, FRAP assay has been used widely to study the antioxidant capacity of pure compounds, foods and beverages [68,69]. The reaction measures the reduction of ferric 2,4,6-tripyridyl-s-triazine (TPTZ) complex to a colored product induced by polyphenols at pH 3.6. The antioxidant capacity is expressed as generated Fe2+ at a fixed time (usually 6 minutes).
The FRAP value of green and black tea has been reported by several investigators [20,70,71].Benzie et al. [70] reported that the FRAP index of green tea is higher than that of black tea. However, as was shown by Benzie et al. [70], high variations of the results between different brands were observed. Interestingly, based on FRAP results it has been proposed that a cup of green tea have a similar antioxidant capacity than 100-200 mg of ascorbic acid.
Then, a consumption of several cups of green tea per day would offer the same antioxidant potential (FRAP) as almost 1 gram of vitamin C.
CUPRAC Assay
CUPRAC assay, proposed originally by Apak et al. [72], is based on the reduction of the complex copper(II)-neocuproine to copper(I)-neocuproine by the combined action of all antioxidants (reducing agents) present in the sample. The complex copper(I)-neocuproine has a characteristic visible band at 450 nm, being the absorption band measured to estimate the
antioxidant capacity of the sample. Apak et al. [73] reported that the CUPRAC value of green tea was among the higher values of several herbal infusions. Furthermore, they reported that ascorbic acid increase the antioxidant capacity of green tea, being the CUPRAC value in presence of lemon 1.7 times higher than that of green tea alone. In this context, similar results of ascorbic acid contribution to TEAC values have been found by Majchrzak et al. [74].
Limitations
The beneficial effects of green tea on the human health have been established in several studies [10,11]. Nevertheless, the published data of the in vitro scavenging activity of green tea involve both different methodologies and experimental procedures. In addition, different values have been reported depending on the commercial brand of the green tea used [70].
Therefore, a comprehensive understanding of the in vitro antioxidant capacity of green tea is difficult and requires an exhaustive analysis of the methodology used and the experimental condition employed in each work. These aspects are stressed in the data of Table 1. As can be seen in Table 1, the antioxidant activity of green tea depends on the methodology used and the procedure of the extraction. Furthermore, within a single assay, the values are dependent of the green tea brand, i.e. in Table 1 using ORAC-PE, LDL, and FRAP assays high fluctuations of the antioxidant capacity values have been published for different green tea brands. In addition, the results obtained using a particular method not always are expressed in the same units, making difficult the comparison of results. For example, employing DPPH test, the results have been reported in terms of IC50, DPPH consumed in percentage or in base of epigallo-catechin-gallate (EGCG) equivalents. In addition, in the analysis of the state of the art, it is necessary to taken into account that the methodologies provide different information. In particular, it must be considered that a given index can be an indication of the total amount of antioxidants (without a discrimination regarding their reactivity, TEAC, DPPH, TRAP, ORAC-FL, FRAP), or be an indication of the quantity and reactivity of the antioxidants present in green tea sample (ORAC-PGR). Moreover, it should be considered that each antioxidant capability assay has their own advantages and limitations.
The simplicity to carry out procedures based on the bleaching of ABTSy+ and DPPH, is one of the most important advantage of these assays. However, the free radicals involved (ABTSy+ and DPPH) are very far from those free radicals relevant in oxidative stress situations. The analysis of the kinetics obtained by ABTSy+ assay is complex [75-77] and can be computed as antioxidant compounds that are pro-oxidant in biological systems, such as hydrogen peroxide or organic hydroperoxides [78]. Furthermore, when the consumption of ABTSy+ is measured at a single (long) reaction time, the result obtained is related to stoichiometric factors and gives no information regarding the reactivity of the tested compounds. The interpretation of data obtained by employing DPPH as the stable free radical is less straightforward. When applied to a fixed time at a pure compound, the method provides stoichiometric factors for highly reactive compounds and/or mixtures, and provides reactivity and stoichiometric factors for low reactivity compounds [79,80].
Table 1. Antioxidant capacity values of green tea evaluated by different methodologies
Method Value Units Experimental conditions Reference TEAC 6.0 mM Trolox eq /L Boiling water (5 min) [18]
a Estimated using a DPPH concentration of 0.1mM, and a concentration of dry green tea extract of 25 µg. b DPPH concentration = 60 µM. c DPPH concentration = 0.2 mM, and a concentration of dry green tea extract = 0.4 mg/mL. d DPPH concentration = 20 mg/mL, and a concentration of green tea extract = 250 µL/mL. c DPPH concentration = 0.25 mM, and a concentration of dry green tea extract = 50 µg/mL.f Lag times obtained with a LDL concentration of 80 µg of cholesterol/mL, and a green tea infusion concentration of 1 µL/mL.
On the other hand, procedures based on the evaluation of the protection given by antioxidants to a target molecule being oxidized by free radicals provide information related to stoichiometric factors and/or reactivity. The TRAP assay is based on the estimation of lag times. Therefore, the index derived from this procedure is only determined by stoichiometric factors. In fact, it only provides an indication of the number of radicals trapped per each antioxidant molecule introduced into the system [81]. The ORAC methodology is a procedure based on the estimation of the area under curve of the protective kinetic profiles. Therefore, the index is related with the origin of the area under curve. Thus, if the target molecule is protected totally by the antioxidants (ORAC-FL), the area under the curve and, in consequence, the ORAC-index is mostly governed by the stoichiometry of the reaction.
However, if the area under curve is related only with a decrease in the initial rate of the target molecule consumption (ORAC-PGR), the ORAC index would reflex the reactivity of the additives. Similarly, protection of LDL is a complex system, and is necessary to consider the initiator of the LDL oxidation for the analysis of the results. If copper is used, it must be
considered the possible chelating effect of the additives. If AAPH is employed as initiator, the delay of the LDL peroxidation chain could be determined by the interaction of the antioxidants with the primary radicals (peroxyl radicals) or with a chain-breaking antioxidant capacity. On the other hand, a disadvantage of LDL assay is that the LDL particles are very different depending of the subject, or even of the diet. Then, the reproducibility, and the comparison of results is not always warranted.
Ferric and cupric reducing power assays (FRAP and CUPRAC) have the limitation that they are only titrating all the antioxidants and those molecules with reducing power towards ferric or cupric complexes. In addition, a shortcoming of FRAP methodology is that the pH used is very different to the physiological value.
Regarding the validity of these indexes as indicators of the quality of a beverage and/or as useful indicators of pathological situations, there are two questions:
• Is possible to estimate the antioxidant capacity of green tea employing only one assay? and
• Is there any relationship between the measured index in a food or beverage and the impact it has on the antioxidant capacity status of the organism?
As expected, there is not a clear-cut yes-or-not answer to these broad questions and it strongly depends on the system considered. For example, using a DPPH-based method [24] it was found that green tea was 6.5 times more powerful as an antioxidant capacity than black tea. However, in preventing the lipid peroxidation of renal homogenates induced by hydrogen peroxide and Fe(II), green tea was only 1.5 time more efficient. Evidently, other factors, such as the distribution and metal chelating characteristics, preclude a quantitative relationship between the amount of antioxidants present in a food or beverage and their biological effect.
In in vivo evaluations, the relationship between ingest and observed levels is still more indirect, since factors such as absorption at the gut level and metabolization can be completely different for the different antioxidants present in the samples.
Considering the aspects above discussed should be recommendable that for the evaluation of the in vitro antioxidant capacity of foods or beverages should be used similar experimental conditions and as many assays as possible. Recently, Seeram et al. [82] have studied the antioxidant capacity of beverages commonly consumed in United States.
Interestingly, the study was developed considering five in vitro antioxidant capacity measuring methodologies (DPPH, ORAC-FL, FRAP, TEAC, and LDL) and different green tea brands. This allows obtaining a more complete profile of the antioxidant capacity, and to correlate the results of different samples. Depending of the assay and the brand, iced green tea showed higher or similar antioxidant ability than iced black and white teas.
3. C
ONCLUSIONGreen tea antioxidant potential has been measured by a large number of methodologies.
All studies point to a high charge of antioxidants, mostly phenolic compounds. Comparison with other infusions, particularly black tea, is difficult since the reported values are strongly influenced by the employed methodology. The data discussed in the present review
emphasized two points: i) ranking of infusions or beverages can be proposed only when the same methodology is employed, both the same extraction procedure and the assay employed to evaluate the antioxidant capacity, and ii) to assess the antioxidant capacity of a infusion, several methodologies should be considered, taking into account the factors that condition the measured index (either the amount of antioxidants and/or their reactivity in the free radical scavenging processes).
A
CKNOWLEDGMENTSThis work was supported by FONDECYT n°11060323 and 1070285.
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EFERENCES[1] Lambert, JD; Hong, J; Yang, GY; Liao, J; Yang, CS. Inhibition of carcinogenesis by polyphenols: evidence from laboratory investigations. The American Journal of Clinical Nutrition, 2005,81,284S–291S.
[2] Yang, CS; Lambert, JD; Hou, Z; Ju, J; Lu, G; Hao, X. Molecular targets for the cancer preventive activity of tea polyphenols. Molecular Carcinogenesis, 2006,45,431–435.
[3] Higdon, JV; Frei, B. Tea catechins and polyphenols: health effects, metabolism, and antioxidant functions. Critical Reviews in Food Science and Nutrition, 2003,43,89–43.
[4] Weisburger, JH. Tea and health: the underlying mechanisms. Proceedings of the Society for Experimental Biology and Medicine, 1999,220,271–275.
[5] Hertog, ML; Feskens, EM; Hollman, PH; Katan, MB; Kromhout, D. Dietary flavonoids and cancer risk in the zutphen elderly study. Nutrition and Cancer, 1994,22,175-184.
[6] Yang, CS; Wang, ZY. Tea and cancer. Journal of the National Cancer Institute, 1993,85,1038-1049.
[7] Hollman, PH; Hertog, ML; Katan, MB. Role of dietary flavonoids in protection against cancer and coronary heart disease. Biochemical Society Transactions, 1996,24,785-789.
[8] Wang, ZY; Huang, MT; Lou, YR; Xie, JG; Reuhl, KR; Newmark, HL; Ho, CT; Yang, CS; Conney, AH. Inhibitory effects of black tea, green tea, decaffeinated black tea, and decaffeinated green tea on ultraviolet-b light induced skin carcinogenesis in 7,12-dimethylbenz[a]anthracene-initiated skh-1 mice. Cancer Research, 1994,54,3428-3435.
[9] Lambert, JD; Sang, S; Yang, Ch. Biotransformation of green tea polyphenols and the biological activities of those metabolites. Molecular Pharmaceutics, 2007,4,819–825.
[10] Cabrera, C; Artacho, R; Giménez, R. Benefical effects of green tea-A review. Journal of the American College of Nutrition, 2006,25,79-99.
[11] Mckay, DL; Blumberg, JB. The role of tea in human health: An update. Journal of the American College of Nutrition, 2002,21,1-13.
[12] USDA: “USDA Database for the flavonoid contents of selected foods” Betsville: US Department of Agriculture, 2003.
[13] Vinson, J; Dabbagh, Y; Serry, M; Jang, J. Plant flavonoids, especially tea flovanols, are powerful antioxidants using in vitro oxidation model for heart disease. Journal of Agricultural and Food Chemistry, 1995,43,2800-2802.
[14] Miller, NJ; Rice-Evans C; Davies, MJ; Gopinathan, V; Milner A. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates.Clinical Science, 1993,84,407-412.
[15] Strube, M; Haenen, GR; Van Den Berg, H; Bast, A. Pitfalls in a method for assessment of total antioxidant capacity. Free Radical Research, 1997,26,515-521.
[16] Henriquez, C; Aliaga, C; Lissi, E. Formation and decay of the ABTS derived radical cation: A comparison of different preparation procedures. International Journal of Chemical Kinetics, 2002,34,659-665.
[17] Re, R; Pellegrini, N; Proteggente, A; Pannala, A; Yang, M; Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology & Medicine, 1999,26,1231-1237.
[18] Pellegrini, N; Serafini, M; Colombi, B; Del Rio, D; Salvatore, S; Bianchi, M; Brighenti, F. Total antioxidant capacity of plant foods, beverages and oils consumed in Italy assessed by three different in vitro assays. The Journal of Nutrition, 2003,133,2812-2819.
[19] Miller, N; Castelluccio, C; Tijburg, L; Rice-Evans, C. The antioxidant properties of theaflavins and their gallate esters-radical scavengers or metal chelators? FEBS Letters, 1996,392,40-44.
[20] Rusak, G; Komes, D; Likic, S; Horzic, D; Kovac, M. Phenolic content and antioxidative capacity of green and white tea extracts depending on extraction conditions and the solvent used. Food Chemistry, 2008,110, 852–858.
[21] Manian, R; Anusuya, N; Siddhuraju, P; Manian, S. The antioxidant activity and free radical scavenging potential of two different solvent extracts of Camellia sinensis (L.) O. Kuntz, Ficus bengalensis L. and Ficus racemosa L. Food Chemistry, 2008,107,1000–1007.
[22] Bramati, L; Aquilano, F; Pietta P. Unfermented Rooibos tea: Quantitative characterization of flavonoids by HPLC-UV and determination of the total antioxidant activity. Journal of Agricultural and Food Chemistry, 2003,51, 7472-7474.
[23] Brand-Williams, W; Cuvelier, ME; Berset, C. Use of a free radical method to evaluate antioxidant activity. Food Science and Technology, 1995,28,25-30.
[24] Yokozawa, T; Dong, E; Nakagawa, T; Kashiwagi, H; Nakagawa, H; Takeuchi, S;
Chung, HY. In vitro and in vivo studies on the radical-scavenging activity of tea.
Journal of Agricultural and Food Chemistry, 1998,46,2143-2150.
[25] Katsube, T; Tabata, H; Ohta, Y; Yamasaki, Y; Anuurad, E; Shiwaku, K; Yamane, Y.
Screening for antioxidant activity in edible plant products: comparison of low-density lipoprotein oxidation assay, DPPH radical scavenging assay, and Folin-Ciocalteu assay.
Journal of Agricultural and Food Chemistry, 2004,52,2391-2396.
[26] Gow-Chin, Y; Chen, HY. Antioxidant activity of various tea extracts in relation to their antimutagenicity. Journal of Agricultural and Food Chemistry, 1995,43,27-32.
[27] Bastos, DH; Saldanha, LA; Catharino, RR; Sawaya, AC; Cunha, IB; Carvalho, PO;
Eberlin, MN. Phenolic antioxidants identified by ESI-MS from Yerba mate (Ilex paraguariensis) and green tea (Camelia sinensis) extracts. Molecules, 2007,12,423-432.
[28] Satoh, E; Tohyama, N; Nishimura, M. Comparison of the antioxidant of roasted tea with green, oolong, and black teas. International journal of Food Sciences and Nutrition, 2005,56,551-559.
[29] Chen, Ch; Tang, HR; Sutcliffe, L; Belton, P. Green tea polyphenols react with 1,1-diphenyl-2-picrylhydrazyl free radicals in the bilayer of liposomes: direct evidence from electron spin resonance studies. Journal of Agricultural and Food Chemistry, 2000,48,5710-5714.
[30] Cao, G; Sofic, E; Prior, RL. Antioxidant capacity of tea and common vegetables.
Journal of Agricultural and Food Chemistry, 1996,44,3426-3431.
[31] Prior, RL; Cao, G. Antioxidant capacity and polyphenolic components of teas:
implications for altering in vivo antioxidant status. Proceedings of the Society for Experimental Biology and Medicine, 1999,220,255-261.
[32] Brownmiller, C; Howard, LR; Prior, RL. Processing and storage effects on monomeric anthocyanins, percent polymeric color, and antioxidant capacity of processed blueberry products. Journal of Food Science, 2008, 73,H72-H79.
[33] Niki, E. Free radical initiators as source of water - or lipid-soluble peroxyl radicals.
Methods in Enzymology, 1990,186,100-108.
[34] Cao, G; Alessio, HM; Cutler, RG. Oxygen-radical absorbance capacity assay for antioxidants. Free Radical Biology & Medicine, 1993,14,303-311.
[35] Henning, SM; Fajardo-Lira, C; Lee, HW; Youssefian, A; Go, V; Heber, D. Catechin content of 18 teas and a green tea extract supplement correlates with the antioxidant capacity. Nutrition and Cancer, 2003,45,226–235.
[36] Caldwell, Ch. Oxygen radical absorbance capacity of the phenolic compounds in plant extracts fractionated by high-performance liquid chromatography. Analytical Biochemistry, 2001,293,232–238.
[37] Chandra, S; Gonzalez de Mejia, E. Polyphenolic compounds, antioxidant capacity, and quinone reductase activity of an aqueous extract of Ardisia compressa in comparison to mate (Ilex paraguariensis) and green (Camellia sinensis) teas. Journal of Agricultural and Food Chemistry, 2004, 52,3583-3589.
[38] Ou, B; Hampsch-Woodill, M; Prior, RL. Development and validation of an improved oxygen radical absorbance capacity assay using fluorescein as the fluorescent probe.
Journal of Agricultural and Food Chemistry, 2001,49,4619-4626.
[39] Bisby, RH; Brooke, R; Navartman, S. Effect of antioxidant oxidation potential in the oxygen radical absorption capacity (ORAC) assay. Food Chemistry, 2008,108,1002-1007.
[40] López-Alarcón, C; Lissi, E. A novel and simple ORAC methodology based on the interaction of pyrogallol red with peroxyl radicals. Free Radical Research, 2006,40,979-985.
[41] López-Alarcón, C; Lissi, E. Interaction of pyrogallol red with peroxyl radicals. A basis for a simple methodology for the evaluation of antioxidant capabilities. Free Radical Research, 2005,39,729-736.
[42] Omata, Y; Saito, Y; Yoshida, Y; Niki, E. Simple assessment of radical scavenging capacity of beverages. Journal of Agricultural and Food Chemistry, 2008,56,3386–
3390.
[43] Alarcón, E; Campos, AM; Edwards, AM; Lissi, E; López-Alarcón, C. Antioxidant capacity of herbal infusions and tea extracts. A comparison of ORAC-fluorescein and ORAC-pyrogallol red methodologies. Food Chemistry, 2008,107,1114-1119.
[44] Prior, RL; Hoang, H; Gu, L; Wu, X; Bacchiocca, M; Howard, L; Hampsch-Woodill, M;
Huang, D; Ou, B; Jacob, R. Assays for hydrophilic and lipophilic antioxidant capacity
(oxygen radical absorbance capacity (ORAC-FL)) of plasma and other biological and food samples. Journal of Agricultural and Food Chemistry, 2003,51,3273-3279.
[45] Oxygen radical absorbance capacity (ORAC) of selected foods – 2007. Nutrient data laboratory Beltsville Human Nutrition Research Center (BHNRC). Agricultural Research Service (ARS) U.S. Department of Agriculture (USDA).
[46] Cao, G; Booth, SL; Sadowski, JA; Prior, RL. Increases in human plasma antioxidant capacity after consumption of controlled diets high in fruit and vegetables. The American Journal of Clinical Nutrition, 1998,68,1081–1087.
[47] Rietveld, A; Wiseman, S. Antioxidant Effects of tea: evidence from human clinical trials. The Journal of Nutrition, 2003,133,3285S-3292.
[48] Wayner, DD; Burton, GW; Ingold, KU; Locke, S. Quantitative measurement of the total peroxyl radical-trapping antioxidant capability of human blood plasma by controlled lipid peroxidation. FEBS Letters, 1985,187,33-37.
[49] Lissi, E; Salim-Hanna, M; Pascual, C; Del Castillo, MD. Evaluation of total antioxidant potential (TRAP) and total antioxidant reactivity from luminol-enhanced chemiluminescence. Free Radical Biology & Medicine, 1995,18,153-158.
[50] Bunkova, R; Marova, I; Nemec, M. Antimutagenic properties of green tea. Plant Foods for Human Nutrition, 2005,60,25–29.
[51] Pietta, PG; Simonetti, P; Gardana, C; Brusamolino, A; Morazzoni, P; Bombardelli, E.
Catechin metabolites after intake of green tea infusions. Biofactors, 1998,8,111-118.
[52] Pietta, PG; Simonetti, P; Gardana, C; Brusamolino, A; Morazzoni, P; Bombardelli, E.
Relationship between rate and extent of catechin absorption and plasma antioxidant status. Biochemistry and Molecular Biology International, 1998,46,895-903.
[53] Serafini, M; Ghiselli, A; Ferro-Luzzi, A. In vivo antioxidant effect of green and black tea in man. European Journal of Clinical Nutrition, 1996,50,28-32.
[54] Ross, R. The pathogenesis of atherosclerosis a perpective for the 1990s. Nature, 1993,362,801-809.
[55] Brown, J; Khodr, H; Hider, R; Rice-Evans, C. Structural dependence of flavonoid interactions with Cu2+ ions: implications for their antioxidant properties. Biochemical Journal, 1998,330,1173-1178.
[56] Prior, RL; Wu, X; Schaich, K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. Journal of Agricultural and Food Chemistry, 2005,53,4290-4302.
[57] Gieseg, S; Esterbauer, H. Low density lipoprotein is saturable by pro-oxidant copper.
FEBS Letters, 1994,343,188-194.
[58] Thomas, MJ; Chen, Q; Franklin, C; Rudel, LL. A comparison of the kinetics of low-density lipoprotein oxidation initiated by copper or by azobis (2-amidinopropane). Free Radical Biology & Medicine, 1997,23,927-35.
[59] Richelle, M; Tavazzi, I; Offord, E. Comparison of the antioxidant activity of commonly consumed polyphenolic beverages (cofee, cocoa, and tea) prepared per cup. Journal of Agricultural and Food Chemistry, 2001,49,3438-3442.
[60] Hodgson, J; Puddey, I; Croft, K; Burke, V; Mori, T; Caccetta, R; Beilin, L. Acute effects of ingestion of black and green tea on lipoprotein oxidation. The American Journal of Clinical Nutrition, 2000,71,1103–1107.
[61] Miura, Y; Chiba, T; Miura, S; Tomita, I; Umegaki, K; Ikeda, M; Tomita, T. Green tea polyphenols (flavan 3-ols) prevent oxidative modification of low density lipoproteins:
an ex vivo study in humans. The Journal of Nutritional Biochemistry, 2000,11,216–
222.
[62] Tinahones, FJ; Rubio, MA; Garrido-Sánchez, L; Ruiz, C; Gordillo, E; Cabrerizo, L;
Cardona, F. Green tea reduces LDL oxidability and improves vascular function.
Journal of the American College of Nutrition, 2008,27,209-213.
[63] Princen, HMG; Van Duyvenvoorde, W; Buytenhek, R; Blonk, C; Tijburg, LBM;
[63] Princen, HMG; Van Duyvenvoorde, W; Buytenhek, R; Blonk, C; Tijburg, LBM;