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ISSN : 1229-1153(Print)
ISSN : 2465-9223(Online)
Journal of Food Hygiene and Safety Vol.32 No.6 pp.460-469

Comparison of Anti-oxidative Activity in a Single Serving Size of the Commercial Coffees and Teas

Tae-Hun Kim1,2,*, Seulgi Lee3, Jin Woo Seo1, Sun Hye Bing1, Jong Im Kim1, Eui-Ra Kwon1, Gune-Hee Jo1, Jae-Myean Lee1, Joon Sig Choi3*
1Food Analysis Division, Daejeon Metropolitan City Institute of Health and Environment, Daejeon, Korea
2Institute of Biotechnology, Chungnam National University, Daejeon, Korea
3Department of Biochemistry, Chungnam National University, Daejeon, Korea
Correspondence to: Tae-Hun Kim, Food Analysis Division, Daejeon Metropolitan City Institute of Health and Environment, 407, Daehak-ro, Yuseong-gu, Daejeon 34142, Korea 82-42-270-6812,
Correspondence to: Joon Sig Choi, Department of Biochemistry, Chungnam National University, 99, Daehak-ro, Yuseong-gu, Daejeon 34134, Korea 82-42-821-5489,
20170829 20170910 20171013


The aim of this work was to study the comparison of anti-oxidative activity in a single serving size of commercial coffees and teas. Commercial regular coffees and teas, including, brand regular coffees (BCA, BCB, BCC, BCD, and BCE), green tea (GTA, GTB, GTC, and GTD), black tea (BTA, BTB, and BTC), pu-erh tea (PTA, PTB, and PTC), chamomile tea (CTA, CTB, and CTC), peppermint tea (PA, PB, and PC), polygonatum odoratum tea (POTA, POTB, and POTC), and jujube tea (JTA, JTB, and JTC) were assayed for the levels of ascorbic acid, caffeine, total content of polyphenols and flavonoids, and ability to scavenge free radicals, using two in vitro antioxidant assays. The scavenging abilities of BCA and BCC were 664.91 ± 48.87 mg ascorbic acid equivalent/serving size and 624.36 ± 16.18 mg ascorbic acid equivalent/serving size, respectively. The four beverage samples (BCA, BCC, GTD, and BTA) significantly reduced the production of reactive oxygen species (ROS) and intracellular oxidative stress induced by H2O2. These results suggest that the beverages possess significant radical scavenging ability, which may be due to the presence of antioxidants. Furthermore, the significant reducing level of ROS evidences the potential antioxidant effects of these beverages in human cells.


    Chungnam National University

    Free radicals are atoms, molecules, or ions with unpaired electrons. These unpaired electrons are highly reactive towards other substances. Thus, free radicals are highly reactive, and lead to uncontrolled reactions that damage macromolecules, such as proteins, lipids, and DNA. There are several different types of free radicals, derived from oxygen and nitrogen, formed in human body. The oxygen-derived free radicals (referred to as reactive oxygen species, ROS) include the hydroxyl radical (OH), peroxyl radical (ROO), alkoxyl radical (RO), and the superoxide anion (O2•−). The reactive oxygen species (ROS) are continuously generated in human body. For example, ROS are produced as by-products of mitochondrial respiration and processes mediated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, xanthine oxidase, and uncoupled NO synthases1). The term “antioxidant” refers to any molecule capable of stabilizing or deactivating free radicals before they attack cells2). Normally, humans have an antioxidant system, consisting of enzymatic and nonenzymatic components, to protect the cells and organs of the body against the damage caused by free radicals. The enzymatic antioxidants include superoxide dismutase, catalase, and glutathione peroxidase; the nonenzymatic antioxidants involve vitamin C, vitamin E, carotenoids, natural flavonoids, and other compounds3). However, an over-production of ROS radicals leads to an imbalance between the generation and elimination of ROS radicals in the body, leading to oxidative stress4). Oxidative stress is a causative factor in various diseases, such as agerelated degenerative conditions, cancers, cardiovascular diseases, asthma, decline of the immune system, brain dysfunction, liver injury, type 2 diabetes, and the aging process5).

    For those reasons, it is important to find agents that can reduce oxidative stress by directly scavenging free radicals. Several studies report that active dietary ingredients, such as phytochemicals, protect our cells from damage caused by free radicals. These phytochemicals include vitamins C, D, and E, alkaloids (caffeine and theobromine), and polyphenols 6). Vitamin C (ascorbic acid) is a water-soluble free radical scavenger that reduces the levels of ROS radicals and acts as primary defense against aqueous radicals in the blood7). Caffeine, and its metabolites in humans, may be highly effective against lipid peroxidation induced by reactive oxygen species8). Polyphenols and phenolic compounds are ubiquitous in the plant kingdom. More than 8,000 phenolic compounds have been isolated in a wide variety of forms, all possessing one common structural feature: a phenol, which is an aromatic ring bearing at least one hydroxyl substituent 9). Because of their antioxidant activity, polyphenols are useful in the prevention of, and symptomatic relief in, such disorders as neurodegenerative and cardiovascular diseases, cancer, and stroke10). Flavonoids are a family of polyphenols with strong antioxidant activity in humans. Regular intake of flavonoid-rich foods is associated with a delay in the onset of Alzheimer’s disease and a reduction in the risk of developing Parkinson’s disease11).

    The consumption of coffee and tea significantly exceeds that of beer, wine, and soft drinks worldwide12). Aside from water, coffee and tea (green tea, chamomile tea, peppermint tea, polygonatum odoratum tea, pu-erh tea, black tea, and jujube tea) are the most widely consumed beverages in the Asian regions. Most coffee or tea consumers do not drink it for their health13,14). However, coffee and green tea contain a multitude of antioxidants, such as vitamins, caffeine, and polyphenols15). Hence, we assessed the content of ascorbic acid, caffeine, total polyphenols, and flavonoids in a single serving size of the commercial regular coffees and teas.

    Materials and Methods

    Chemicals and reagents

    Ascorbic acid, gallic acid, quercetin, Folin-Ciocalteu’s phenol reagent, aluminum nitrate nonahydrate, potassium persulfate, DPPH (2, 2-diphenyl-1-picrylhydrazyl), ABTS (2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt), MTT (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide), DMSO (anhydrous dimethyl sulfoxide), and Hydrogen peroxide (H2O2) were all obtained from Sigma-Aldrich (Steinheim, Germany). Caffeine, potassium dihyrogen phosphate, trifluoroacetic acid, metaphosphoric acid, sodium carbonate anhydrous, and potassium acetate were purchased from Wako (Osaka, Japan). Acetonitrile, ethanol, and methanol were purchased from Merck (Darmstadt, Germany). DMEM (Dulbecco’s modified Eagle’s medium) and 100 × antibiotic-antimycotic reagent were purchased from Thermo Fisher Scientific (Waltham, MA, USA). FBS (Fetal bovine serum) and H2DCF-DA (Dichlorodihydrofluorescein diacetate) were from Invitrogen Molecular Probes (Eugene, OR, USA). HeLa cell lines were obtained from Korean Cell Line Bank.

    Samples and preparation

    Samples of commercial regular coffee and tea were purchased at nationwide coffee chain stores, and department stores, large discount stores, in Daejeon metropolitan area, Republic of Korea. The commercial brands (A, B, C, D, and E) of regular coffee, used in this study, were purchased in the quantities of three cups per brand (Table 1). The average capacity (in mL) of a cup of each brand of regular coffee (Americano) was A (284 ± 5), B (319 ± 2), C (304 ± 6), D (301 ± 2), and E (325 ± 5). Twenty kinds of leached tea (four types of green tea, three types of black tea, three types of pu-erh tea, three types of chamomile tea, three types of peppermint tea, three types of polygonatum odoratum tea, and one type of jujube tea), and two kinds of solid-extracted tea (two types of jujube tea), were purchased (Table 2). The infusions were prepared by pouring 120 mL of boiled water at 100°C on one tea bag and brewing for 10 min. To analyze cytotoxicity and the levels of reactive oxygen species (ROS), brand regular coffees and aqueous tea extracts were lyophilized using a FreeZone Plus 2.5 freeze dryer (Labconco, MO, USA) and stored at −20°C until analysis.

    Determination of ascorbic acid and caffeine contents in commercial regular coffee and tea extracts by high performance liquid chromatography (HPLC)

    The ascorbic acid and caffeine were determined using chromatographic system equipped with analytical HPLC unit model Nanospace SI-2 system (Shiseido Fine Chemicals, Tokyo, Japan) consisting in a 3201 pump, 3202 degasser, Accela PDA detector, 3004 column oven and 3023 auto sampler. The separation of the analytes was carried out using reversed-phase Phenomenex Kinetex column packed with C18 (5 μm particle size, 250 × 4.6 mm). Ascorbic acid was separated by isocratic elution with water-trifluoroacetic acid (99:1, v/v) as the mobile phase. Caffeine was separated using an isocratic elution with acetonitrile-potassium dihydrogen phosphate (10:90). Chromatograms were recorded at 254 nm for ascorbic acid and 274 nm for caffeine.

    Determination of total polyphenols and total flavonoids

    The total polyphenol contents in the coffee or tea extract were determined by using the Folin-Ciocalteu reagent according to the colorimetric method described by Singleton and Rossi16). Briefly, the reaction mixture was composed by 25 μL of coffee or tea extract, 1.6 mL of deionized water, 75 μL of Folin-Ciocalteu reagent and 0.3 mL of 2% sodium carbonate, placed in microtubes. The microtubes were agitated, held for 1 hr, and the absorbance was measured at 700 nm with a Cary 300 UV-visible spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). The total polyphenol contents were expressed as mg gallic acid equivalent (GAE) per serving size. In addition, total flavonoids in the coffee or tea extract were estimated using the colorimetric assay previously described by Chang et al. with some modifications17). A volume of 50 μL of each the coffee or tea extract was added in a tube and subsequently, a sequential addition of 450 μL ethanol at 80%, 100 μL aluminum nitrate at 10%, 100 μL potassium acetate (1 M), and 4.3 mL ethanol at 80% to each extract sample was performed. Samples were maintained during 40 min in the dark at room temperature. The absorbance of the mixture was then measured at 415 nm against a blank of deionized water. The content of total flavonoids was expressed as mg quercetin equivalent (QE) per serving size.

    Antioxidant capacity

    The DPPH•+ assay for antioxidant activity was conducted by the method of Gyamfi et al. with slight modifications18). Briefly, coffee or tea extract (10 μL) were mixed with DPPH•+ (2,990 μL; 0.4 mM) followed by incubation in the dark at room temperature for 10 min. Absorbance at 520 nm was measured against deionized water as blank using a Cary 300 UV-visible spectrophotometer. All results were expressed as mg ascorbic acid equivalent (AE) per serving size. The antioxidant activity measured with ABTS was carried out according to the method described by Re et al. with some modifications19). ABTS•+ was generated by reacting an ABTS aqueous solution (7.4 mM) with K2S2O8 (2.6 mM, final concentration) in the dark for 24 hr and adjusting the Abs 734 nm to 3.1 with water. 0.05 mL of coffee or tea extract was added to 9.95 mL ABTS•+ solution and the absorbance were measured at 734 nm after 90 min. Results were expressed as mg ascorbic acid equivalent (AE) per serving size.

    Cytotoxicity assay

    HeLa cells, human cervical cancer cells, was cultured in DMEM with 10% FBS, 1% antibiotic-antimycotic agent. Cell lines were maintained in an incubator (5% CO2, 95% relative humidity, 37°C). Evaluation of cytotoxicity was performed by the MTT assay in HeLa cells. Cells were seeded at a density of 2 × 104 cells/well in 96-well plate and were incubated for 1 day before adding the coffee or tea extract. Cells were treated with brand coffee (BCA and BCC), green tea D, and black tea A at 1/8 to 1 serving size concentrations for 24 hr at 37°C. Then, 26 μL of MTT stock solution (2 mg/mL) was added and incubated for 4 hr at 37°C. MTT-containing medium was removed and 150 μL DMSO was added to each well to dissolve the formazan crystal formed by live cells. The absorbance of each well was read on a microplate reader (VersaMax, Molecular Devices, US) at 570 nm.

    Intracellular ROS level

    Intracellular ROS level was determined by using fluorescent probe H2DCF-DA20). HeLa cells were seeded in 96- well plates at 1 × 104 cells/well. To evaluate the direct effect, 24 hr after seeding, exposed for 24 hr to brand coffee (BCA and BCC), green tea D, and black tea A at 1/8 to 1 serving size concentrations. Afterwards, 100 μL of H2DCF-DA (100 μM) in serum- and phenol red-free DMEM was added to each well for 30 min at 37°C, and cells were washed once with PBS. To test the protective effect against oxidative stress, cells were pretreated during 24 hr with brand coffee (BCA and BCC), green tea D, and black tea A at 1/8 to 1 serving size concentrations, the H2DCF-DA (100 μM) probe was added to each well for 30 min at 37°C, and the cells were washed once with PBS and fresh phenol red-free DMEM containing 500 μM H2O2 was added to all cultures except controls for 10 min at 37°C. In both experiments, intracellular ROS were measured using a HTS Multi Label Reader (Perkin Elmer, Waltham, MA, USA) at excitation and emission wavelengths of 485 and 538 nm, respectively.

    Statistical analysis

    Each parameter was analyzed in triplicate. Results are shown as means ± standard deviations. The results were statistically analyzed by Analysis of variance (ANOVA), and the unpaired t-test. Significance was accepted at p ≤ 0.05. All statistical analyses were performed using the SPSS v.12.0 software package.

    Results and Discussion

    Ascorbic acid, caffeine, total polyphenol, and flavonoid contents of commercial regular coffee and tea

    The values of the ascorbic acid, caffeine, total polyphenol, and flavonoid contents of the beverages are shown in Table 3. Green tea (GTA, GTB, GTC, GTD) and black tea (BTA, BTB, BTC) were not significantly different (p = 0.1000) in their content of ascorbic acid, while other beverages did not contain ascorbic acid. This may be because the roasting of coffee, fermentation of pu-erh tea, and drying of other teas destroy the high content of ascorbic acid originally present in the green coffee bean and tea leaves21). Therefore, we cannot exclude the possibility that the antioxidant capacity of these beverages is produced by caffeine and phenolic compounds, rather than by ascorbic acid, which is easily destroyed by heat, air, and improper storage and processing of foods. Brand coffees and green, black, and pu-erh teas all contain caffeine. The brand coffee A and E had a higher caffeine content (p < 0.05) than other coffees. This result may be related to the different species (BCA, Robusta 20%) of the coffee beans and long extraction time (BCE, 29 seconds) of the espresso. Other studies showed that Robusta coffee extracts contain twice as much caffeine as Arabica22). Weight for weight, tea leaves contain more caffeine than coffee beans; however, a serving size of tea (green, black, or pu-erh tea) contains only 15 to 64 mg caffeine compared with a serving size of brand coffee, which contains between 150 to 203 mg. This is because caffeine is not effectively extracted by brewing, unless the beverage is “stewed”. As shown in Table 2, although the amount of comparison is different, our results showed that the three black teas had a higher caffeine content (p < 0.0001) than green and pu-erh teas in a single serving size of the teas. Black tea typically has more caffeine than do green and pu-erh teas, but the content of caffeine can vary depending on the type of tea, grade of comminution of tea leaves, and maturity of the leaves; young tea leaves have a higher concentration of caffeine than do mature leaves23). All brand coffee samples presented a rich source of phenolic compounds; the total polyphenol content in brand coffees was significantly (p < 0.05) higher than that in the other beverages. Total phenolic content was significantly higher (p < 0.0001) in jujube tea A than those in jujube tea B and jujube tea C. Jujube teas B and C are solid-extract teas that are made from the concentrated extract of the jujube fruit, and various nuts, such as pine nuts and almonds; conversely, jujube tea A is a leached tea. The percentages of jujube in jujube tea A, B, and C were 100%, 1.6%, and 2.2%, respectively. Hence, the high level of total polyphenol in jujube tea A may be attributable to the content of jujube. The green (GTA, GTB, GTC, GTD) and black teas (BTA, BTB, BTC) were not significantly different in their polyphenol and flavonoid contents (p = 0.5490 and p = 0.0571, respectively).

    Antioxidant activity of commercial regular coffee and tea

    The evaluation of antioxidant activity of commercial regular coffees and teas was conducted in vitro by evaluation of DPPH and ABTS radical scavenging ability; the results are shown in Table 4. These methods are widely used to determine in vitro antioxidant activity of foods and beverages 24). The determination of radical scavenging activity using DPPH showed that the brand coffee BCC had the highest scavenging ability (664.91 mg ascorbic acid equivalent/ serving size), while polygonatum odoratum tea (POTB) had the lowest (1.40 mg AE/serving size). This result indicates that the brand coffee BCC and polygonatum odoratum tea (POTB) possess an antioxidant activity equivalent to 664.91 mg and 1.40 mg of ascorbic acid, respectively. The assessment of quenching activity, using DPPH, indicated that the beverages can be ranked in descending order: BCC > BCA > BCE > BCD > BCB > BTA > GTD > GTA > GTB > BTB > BTC > GTC > PB > PTA > PTC > PTB > PC > PA > JTA > CTB > POTA > CTA > CTC > POTC > JTC > JTB > POTB. The antioxidative effectiveness of these beverages is due to the presence of antioxidant compounds, which are mainly polyphenols. As shown in Table 3, the phenolic content was higher in the coffee brand C than in polygonatum odoratum tea B. The assessment of scavenging ability using ABTS indicated that coffee brand C had the highest scavenging ability, with 624.36 mg AE/serving size, while jujube tea (JTB) had the lowest value of 0.20 mg AE/serving size. Jujube tea (JTB) and polygonatum odoratum tea (POTB) were not significantly different in their DPPH and ABTS values (p = 0.8085 and p = 0.4460, respectively). The ABTS antioxidant activity assay showed a similar trend to that observed with the DPPH assay.

    Correlation between the antioxidant compounds and antioxidant activity

    This study aimed to improve the understanding of how antioxidant compounds affect the antioxidative activity of these beverages. The assessment of DPPH and ABTS free radical scavenging ability indicated significant and strongly positive Pearson’s correlation between caffeine and total polyphenol and flavonoid content of these beverages (Table 5). Thus, it can be inferred that caffeine, polyphenols, and flavonoids are important contributors to the antioxidant activity of these beverages. Similarly, the correlation between DPPH and ABTS showed a correlation coefficient that approached 1 (r = 0.9948, p < 0.0001); this is likely because both methods have a common mechanism for eliminating artificially formed free radicals. Contrarily, no linear correlation was confirmed between the concentration of ascorbic acid and DPPH (r = 0.1982, p = 0.3217) or ABTS assay (r = 0.1893, p = 0.3442), possibly because of their low concentration.


    We have shown that among brand regular coffees (Americano) and leached teas, brand coffees A and C (BCA, BCC), and green tea D and black tea A, were those with the highest antioxidant capacity (Table 4). Therefore, we have evaluated the possible antioxidant effect of the four beverages in HeLa cells. First, we examined the cytotoxicity of each beverage in order to choose an adequate concentration of that beverage. Then, we determined the direct effects of each beverage on the level of intracellular ROS using the dichlorofluorescein assay. Finally, we assessed the ability of each beverage to protect against the H2O2-induced increase in intracellular ROS. The cytotoxicity of brand coffees BCA and BCC, as well as green tea D and black tea A, was measured in HeLa cells, after 24 hr of exposure, using the MTT assay (Fig. 1). The concentrations of each residue, corresponding to 1/8 to 1 cup of each beverage sample, are shown in Table 6. After a 24-hr incubation, the viability of HeLa cells was not affected (> 96%) by green tea D and black tea A at the tested concentrations (~1 cup, Fig. 1). Additionally, brand coffee C did not exert any cytotoxic effects at the concentration of 1/2 cup (cell viability~93.2%), while a clearly toxic effect at the concentration of 1 cup was observed for both brand coffees BCA and BCC.

    Effect of commercial regular coffee and tea on the intracellular level of ROS

    After a 24-hr incubation with the beverage samples, the level of intracellular ROS was evaluated in HeLa cells (Fig. 2(A)). All the tested beverage samples dose-dependently induced a relevant decrease in the levels of ROS at the concentrations of 1/8~1 cup. Additionally, all the tested beverage samples induced a highly significant (p < 0.001) decrease in the basal levels of ROS at the concentration of 1/8 to 1 cup (except at 1/8 cup, GTD). In particular, when HeLa cells were treated with 1/2 cup of BCC, the level of ROS decreased by approximately 53% compared with that in the control cells. Overall, the inhibition of ROS production by brand coffees BCA and BCC was greater than that by the leached teas (at 1/8~1/2 cup, p < 0.05). Previous studies found that DPPH and ABTS radical activities of beverages scavenging showed excellent antioxidative effects of beverages due to large amounts of total polyphenols, flavonoids, and caffeine contents. Tables 3 and 4 show that the levels of antioxidant compounds (denoted by the total content of polyphenols, flavonoids, and caffeine) and antioxidative activity of BCA and BCC were higher than those of GTD and BTA; accordingly, the reduction in the level of ROS, induced by of BCA and BCC, was greater than that induced by GTD and BTA (Fig. 2(A)). Therefore, we conclude that the high radical scavenging ability of these beverages effectively suppressed basal ROS production in HeLa cells.

    Protection of commercial regular coffee and tea against oxidative stress by intracellular ROS

    We evaluated whether these beverage samples exerted protective activity against an increase in the level of ROS induced by exposure to 500 μM H2O2 (10 min, 37 ); the results are shown in Fig. 2(B). In the group treated with H2O2, the fluorescence intensity of DCF increased to 135%, compared with 100% observed in the control group. All the tested samples dose-dependently (1/8~1 cup) reduced the intracellular oxidative stress that was induced by H2O2, which generates hydroxyl radicals (p < 0.05). In particular, at the concentration of 1/4 cup, BCA showed 54.53%, which is approximately a 60% reduction in the levels of ROS compared to H2O2. This protective effect paralleled the ability of the beverage samples to reduce the level of ROS. The ability of the beverages to reduce the levels of ROS was ranked as follows: brand coffees > black tea > green tea; the protective effect exhibited the same trend. These results are likely due to the presence of antioxidant compounds, such as ascorbic acid, caffeine, and total polyphenols and flavonoids, in these beverages. This suggests that these beverage samples can alter the oxidative environment of the cells.

    In conclusion, this study revealed that brand regular coffees had the highest antioxidant activity, while green and black tea had adequate antioxidant activity. Among the brand coffees and leached teas, brand coffees BCA and BCC, green tea D, and black tea A were found to have higher antioxidant activity. The substances that contribute to the antioxidant capacity of these beverages are polyphenols, flavonoids, and caffeine, but not ascorbic acid. Additionally, brand coffees BCA and BCC, green tea D, and black tea A showed high free radical scavenging ability in HeLa cells that had been stimulated by oxidative stress. Further experimental and clinical studies on the relevant antioxidant compounds are needed to expand the significance of these results.


    This work was supported by research fund of 2016 Chungnam National University.



    Viability curves of HeLa cells after 24 hr of incubation with beverage samples. Data were obtained with the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Each point indicates the average and standard deviation of three independent experiments.


    Intracellular reactive oxygen species (ROS) level of HeLa cells. (A) Direct effect on intracellular ROS generation treated for 24 hr with different concentrations of beverages. (B) Protective effect of the beverages against hydrogen peroxide (H2O2)-induced production of ROS, evaluated by the dichlorofluorescein assay. Data are expressed as mean ± standard deviation of three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001.


    Commercial regular coffee samples

    Commercial tea samples

    The contents of ascorbic acid, caffeine, and phenolic (total polyphenols and flavonoids) in commercial regular coffees and teas.

    All values are shown as mean ± standard deviation (n = 3)

    Antioxidant activity of the regular coffees and teas evaluated by the ABTS•+ and DPPH•+ assays.

    All values are shown as mean ± standard deviation (n = 3)

    Correlation coefficients of between contents of ascorbic acid, caffeine, and total polyphenols, total flavonoids, and antioxidant activity in a serving size of the commercial regular coffees and teas

    Concentrations of total residues (μg mL–1 dry weight) of tested beverages.

    All values are shown as mean ± standard deviation (n = 3)


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