Accepted Manuscript

Short communication

Modification of the Cellular Antioxidant Activity (CAA) Assay to Study Phe-

nolic Antioxidants in a Caco-2 Cell Line

Mary E. Kellett, Phillip Greenspan, Ronald B. Pegg

PII: S0308-8146(17)31664-3
DOI: https://doi.org/10.1016/j.foodchem.2017.10.035
Reference: FOCH 21858

To appear in: Food Chemistry

Received Date: 10 August 2017

Accepted Date: 8 October 2017

Please cite this article as: Kellett, M.E., Greenspan, P., Pegg, R.B., Modification of the Cellular Antioxidant Activity
(CAA) Assay to Study Phenolic Antioxidants in a Caco-2 Cell Line, Food Chemistry (2017), doi: https://doi.org/
10.1016/j.foodchem.2017.10.035

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 Modification of the Cellular Antioxidant Activity (CAA) Assay

to Study Phenolic Antioxidants in a Caco-2 Cell Line

Mary E. Kellett,a Phillip Greenspanb and Ronald B. Pegg*a

a
Department of Food Science & Technology, College of Agricultural and Environmental

Sciences, The University of Georgia, 100 Cedar Street, Athens, GA, 30602, United States
b
Department of Pharmaceutical & Biomedical Sciences, College of Pharmacy, The University of

Georgia, 250 W. Green Street, Athens, GA, 30602, United States

Running Title: Phenolic Antioxidants and the Cellular Antioxidant Activity Assay

*Corresponding author: Dr. Ronald B. Pegg. Tel: (706) 542-1099. Fax: (706) 542-1050. E-mail:

rpegg@uga.edu

1
Abstract

In vitro assays are widely used to analyze the antioxidant potential of compounds, but they

cannot accurately predict antioxidant behavior in living systems. Cell-based assays, like the

cellular antioxidant activity (CAA) assay, are gaining importance as they provide a biological

perspective. When the CAA assay was employed to study phenolic antioxidants using

hepatocarcinoma (HepG2) cells, quercetin showed antioxidant activity in HepG2 cells; 25 and

250 M quercetin reduced fluorescence by 17.10.9% and 58.62.4%, respectively. (+)-

Catechin, a phenolic antioxidant present in many foods, bestowed virtually no CAA in HepG2

cells. When Caco-2 cells were employed, more robust antioxidant activity was observed; 50 M

(+)-catechin and quercetin reduced fluorescence by 54.11.4% and 63.60.9%, respectively.

Based on these results, likely due to differences in active membrane transport between the cell

types, the Caco-2-based CAA assay appears to be a more appropriate method for the study of

certain dietary phenolics.

Keywords: Antioxidants; Phenolics; Proanthocyanidins (PACs); Catechin; Cell-based Assays;

Caco-2 Cells

2
 1 1. Introduction

2 Multiple in vitro methods of analyzing dietary antioxidants are widely used today, but their

3 overall usefulness has been called into question (Schaich, Tian, & Xie, 2015). This is because the

4 results from in vitro chemical tests under non-physiological conditions simply cannot be

5 extrapolated to predict the performance of the tested sample in biological systems (Lpez-

6 Alarcn, & Denicola, 2013). Most in vitro tests analyze a compounds capability to quench

7 radicals via one of two mechanisms: the hydrogen atom transfer (HAT) method or the single

8 electron transfer (SET) method, which is primarily due to a compounds redox properties (Craft,

9 Kerrihard, Amarowicz, & Pegg, 2012). This oversimplifies the antioxidant action, as in many

10 cases a single system (food or otherwise), or even a single compound, can use both mechanisms

11 concurrently (Migliavacca, Carrupt, & Testa, 1997; Prior, Wu, & Schaich, 2005). Moreover,

12 radical quenching is not the whole role of antioxidant defenses: to protect cells from oxidative

13 stress, antioxidant compounds may up-regulate antioxidant enzymes, modulate gene expression,

14 or change cell signaling (Jones, 2006; Lpez-Alarcn, & Denicola, 2013). Test tube assays,

15 such as oxygen radical absorbance capacity (ORACFL) and ferric reducing antioxidant potential

16 (FRAP), are incapable of examining these additional factors, and for that reason, new methods of

17 antioxidant activity analysis are needed.

18 Cell-based assays are gaining traction as a more biologically relevant technique that serves

19 as a middle ground between in vitro assays and animal feeding studies or human clinical trials.

20 One such assay is the quantitative Cellular Antioxidant Activity (CAA) assay, popularized in the

21 last decade by Wolfe and Liu (2007) although originally conceived and reported by Wang and

22 Joseph (1999). The CAA assay is useful for examining the bioavailability of food antioxidants,

23 because it can take several additional factors into consideration including cellular uptake, and
24 metabolism. Originally, the assay used PC12 cells from the adrenal gland of rats; today HepG2

25 cells, derived from human hepatocellular carcinoma, are reported frequently in the literature

26 (Wolfe & Liu, 2007; Wolfe, Kang, He, Dong, Zhang, & Liu, 2008; Song, Derito, Liu, He, Dong,

27 & Liu, 2010). Though the HepG2 cell line is of human origin, and thus an improvement over

28 PC12 cells, liver cells cannot be the ideal cell for measuring the effectiveness of dietary

29 antioxidants. For this reason, there is a more logical move towards the employment of human

30 colorectal adenocarcinoma (Caco-2) cells; they are of similar morphology, marker enzyme,

31 microvillar structure, tight junction and permeability as small intestinal epithelial cells (Wang &

32 Joseph, 1999). These human colonocytes retain their function in culture; hence, Caco-2 cells are

33 frequently used for modeling the intestinal barrier (Sergent et al., 2005; Wan, Dong, Yu, Sun, &

34 Li, 2015).

35 The CAA assay uses a fluorescent probe (2,7-dichlorofluorescin diacetate or DCFH-DA)

36 to monitor the inhibition of peroxyl radical-induced oxidation inside the cell. The ester form of

37 the dye (DCFH-DA) is employed, because it is nonionic and non-polar and thus can rapidly

38 transport across the cell membrane (LeBel, Ischiropoulos, & Bondy, 1992; Wang & Joseph,

39 1999). Once inside the cell, the DCFH-DA is deacetylated by endogenous cellular esterases and

40 left in the more oxidizable DCFH form (Figures 1 and 2). A free-radical generator, namely 2,2-

41 azobis (2-amidinopropane) dihydrochloride (ABAP), is added to the system and begins to form

42 peroxyl radicals. Other reactive oxygen species (ROS) are generated endogenously through

43 cellular function (Sies, 1993). If an antioxidant is introduced and able to enter the cell, it can

44 compete with radicals and quench them in a variety of ways, keeping the DCFH from being

45 oxidized to form the fluorescent 2,7-dichlorofluorescein (DCF). The principle of the CAA

46 assay, along with possible pathways of radical quenching can be found in Figure 1. The
47 effectiveness of the sum total of these pathways is evaluated by monitoring the intracellular

48 fluorescence resulting from DCFH over a time course: reduced fluorescence compared to a

49 control (i.e., wells void of antioxidant compounds) indicates a sample has substantial antioxidant

50 activity within the cell.

51 The CAA assay is quantitated by the CAA unit (i.e., the relative reduction in fluorescence,

52 expressed in percent), which takes into account relative areas under the curves of sample wells

53 and control wells. The higher the CAA unit, the more effective the antioxidant is in a cellular

54 system. The assay has been used with HepG2 cells for a variety of pure phytochemicals found in

55 food, such as quercetin, gallic acid, and caffeic acid, as well as several common fruit and

56 vegetable extracts literature (Wolfe & Liu, 2007; Wolfe et al., 2008; Song et al., 2010).

57 Commercial products, such as the OxiSelect TM Cellular Antioxidant Assay Kit, are available

58 (Cell Biolabs, 2013). Recently, the assay was modified for analysis in the Caco-2 cell line. Wan

59 et al. (2015) changed cell plating density, probe concentration, and the choice of peroxyl radical

60 generator (i.e., 2,2-azobis(2-methyl propionamidine dihydrochloride, AAPH) for better results.

61 As the assay is still relatively new and is thus continually being modified for a variety of

62 applications, standardization has not yet occurred. In fact, there is no universally accepted

63 method for the measurement of antioxidants (Niki, 2010). For these reasons, discrepancies

64 among labs are common.

65 Previously, CAA assays have been performed for fruit and vegetable extracts using HepG2

66 cells, all with quercetin as the standard (Wolfe et al., 2008; Song et al., 2010; Wan et al., 2015).

67 Other foodstuffs, such as tree nuts, are relatively poor in quercetin, but rich in flavan-3-ols like

68 (+)-catechin, ()-epicatechin, their derivatives, and proanthocyanidins. It is hypothesized that

69 because the intestinal barrier and bioavailability are the main factors affecting the function of
70 antioxidants in vivo, the Caco-2 cell line should serve as a superior model to screen

71 phytochemical-rich foods for potential antioxidant activity. Most food extracts being tested now

72 employ the HepG2 cell line and the quercetin standard curve, which has limitations. In this

73 study, the CAA assay was performed with both HepG2 and Caco-2 cells using both quercetin

74 and (+)-catechin. Thus, the objectives were to assess the CAA assay for the performance of

75 Caco-2 cells in initial stages of antioxidant research and to describe an assay suitable for

76 measuring phenolic antioxidants, such as the flavan-3-ol species abundant in tree nuts.

77

78 2. Materials and methods

79 2.1. Chemicals and supplies

80 2,7-Dichlorofluorescin diacetate or 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA,

81 97% purity), 2,2-azobis(2-amidinopropane) dihydrochloride (ABAP), dimethyl sulfoxide

82 (DMSO), (+)-catechin hydrate (98% purity by HPLC), and quercetin dihydrate (98% purity by

83 HPLC) were purchased from the Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

84 Dulbeccos Modified Eagle Medium (DMEM), Advanced DMEM, Williams Medium E,

85 phosphate-buffered saline (PBS), Hanks Balanced Salt Solution (HBSS), fetal bovine serum

86 (FBS), L-glutamine, 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), insulin,

87 gentamicin, penicillin-streptomycin, and trypsin-EDTA were purchased from Life Technologies

88 (Grand Island, NY, USA).

89

90 2.2. Cell culture

91 Human hepatocellular carcinoma (HepG2) cells were purchased from the American Type

92 Culture Collection (ATCC, Manassas, VA, USA) and cultured via the method described by
 93 Wolfe and Liu (2007). Briefly, cells were cultured in sterile Williams Medium E (WME), along

94 with 5% FBS, 10 mM HEPES, 2 mM L-glutamine, 5 g/mL insulin, and 0.05 g/mL

95 hydrocortisone. Human colon adenocarcinoma (Caco-2) cells were also acquired from the

96 ATCC. Following the method of Xie, Kosiska, Xu, and Andlauer (2013) cells were cultured in

97 Advanced DMEM supplemented with 10% endotoxin-free, heat-inactivated fetal bovine serum

98 (FBS), 1% L-glutamine, 1% penicillin (10,000 U/mL) and 1% streptomycin (10,000 g/mL).

99

100 2.3. Preparation of phenolic test samples

101 Stock solutions of quercetin dihydrate (98% purity by HPLC) and (+)-catechin hydrate

102 (98% purity by HPLC) were prepared in DMSO at a concentration of 50 mg/mL. On the day of

103 analysis, samples were diluted to final concentrations ranging from 5 to 1,000 M in serum-free

104 culture media; the final treatment solutions contained <2% DMSO. A 12.5 mM stock solution of

105 the fluorescent probe DCFH-DA in CH3OH was prepared monthly. Before each experiment,

106 working solutions of 25 M DCFH-DA in serum-free culture media were prepared. A 60 mM

107 ABAP stock solution in HBSS was prepared weekly and diluted to 600 M before experimental

108 use. All DCFH-DA and ABAP solutions were held at -20 C between uses, while samples in

109 DMSO were kept refrigerated at 4 C.

110

111 2.4. Cellular antioxidant activity (CAA) assay

112 Cellular antioxidant measurements were made following the method of Wolfe and Liu (2007)

113 with modifications. After HepG2 or Caco-2 cells reached confluence in the Corning 25-cm2

114 culture flasks, cells were twice washed with sterile PBS (pH 7.4), and then dissociated from the

115 surface using 0.05% trypsin-EDTA. Cells were plated (6.0 104) in 100-L cell culture
116 media/well in Corning Costar 96-well, black, flat bottom tissue culture-treated dishes (VWR

117 International, Suwanee, GA, USA) and incubated until confluent (24 to 48 hours). Confluence

118 was confirmed using the microscope. Wells on the perimeter were left empty to reduce any

119 variation due to plate location. Growth medium was removed after confluence was achieved, and

120 the cells were then washed with PBS to remove any non-adherent and dead cells.

121 Next, 50 L of 25 M DCFH-DA working solution were added to each well, followed by 50

122 L of the antioxidant treatments (in triplicate wells). For a control, 50 L of DCFH-DA and 50

123 L of serum-free culture media (no antioxidant included) were applied to triplicate wells. Once

124 the DCFH-DA and antioxidant treatments were added, the cells were placed in the incubator for

125 1 h at 37 C.

126 After this period, the cells were washed 3 with PBS to ensure that any antioxidant effect

127 observed later in the assay was due only to compounds internalized by the cells. One hundred L

128 of the free radical generator, ABAP (600 M), were then added. The cells were immediately

129 placed in a BMG FLUOstar Omega microplate reader (BMG LABTECH Inc., Cary, NC, USA),

130 where real-time fluorescence was read initially and every five minutes then after for 1 h (i.e., 13

131 readings were read in total). Fluorescence was measured at an excitation wavelength of 485 nm

132 and an emission wavelength of 538 nm.

133

134 2.5. Cell cytotoxicity assay

135 To test the viability of both HepG2 and Caco-2 cell lines after catechin and quercetin

136 treatments were applied (i.e., at concentrations ranging between 2.5 and 250 M), the

137 colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was

138 employed (Gentile, Allegra, Angileri, Pintaudi, Livrea, & Tesoriere, 2012). DCFH-DA and the
139 phenolics were applied to cells in the same fashion as described for the CAA assay and allowed

140 to incubate for 1 h. Then, the cells were thoroughly washed with PBS to remove all phenolic

141 residues and traces of media. Control wells contained MTT and media, but no phenolics. One

142 hundred microliters of serum-free culture media were applied to the cells along with 10 L of 12

143 mM MTT. After 4 h, 85 L of media were removed and 50 L of DMSO were added to each

144 well to dissolve the generated cellular formazan chromagen. The samples were incubated at 37

145 C once more for 10 min and the absorbance of the purple colored formazan was measured at

146 540 nm. The assay was quantitated by comparing the absorbance of samples against that of the

147 control; treatments resulting in significantly (p < 0.05) lower absorbance readings are considered

148 cytotoxic. All assays were performed in triplicate.

149

150 2.6. Quantitation of the CAA assay and statistical analysis

151 Effectiveness of antioxidant treatments for both cell lines was quantitated by examining the

152 percent reduction in fluorescence. Briefly, curves were generated by the 13 fluorescence

153 response readings of each treatment over the course of the 1 h assay. The area under each curve

154 was calculated via integration with the MARS Data Analysis Software (Version 3.20 R2, BMG

155 LABTECH). As expected, control wells exhibited the maximum fluorescence, as there was no

156 inhibition of the ABAP and DCFH-DA reaction. The percent reduction (or the CAA unit) was

157 calculated as follows:

158 CAA units were determined for quercetin and (+)-catechin gathered from triplicate

159 determinations of four separate experiments and reported as meanstandard error.

160
161 3. Results and discussion

162 3.1. CAA results from HepG2 cells

163 The CAA assay was performed using HepG2 cells with concentrations of quercetin ranging

164 from 25 to 250 M. The CAA unit, the index of antioxidant activity, increased in a

165 concentration-dependent manner; the strength of inhibition strongly following a curvilinear

166 pattern as the effect tapered off at higher antioxidant concentrations. Fluorescence was reduced

167 by up to 58.61.0% when in the presence of 250 M quercetin. Our results are in agreement with

168 the methodology and literature recommended in the OxiSelectTM Cellular Antioxidant Activity

169 Kit (Cell Biolabs, Inc., San Diego, CA, USA); the kit recommends using concentrations of

170 quercetin ranging from 31.3 to 2000 M to develop a standard curve. Hofer, Jrgensen, and

171 Olsen (2014) also reported a half-maximal inhibitory concentration (IC50) value for quercetin as

172 71.238.6 M, which again is similar to the values we report as well as those determined using

173 the OxiSelect kit. These findings, however, were quite different from the work of Wolfe and

174 Liu (2007), who popularized the HepG2 version of the assay; they cited CAA values of up to

175 70.0 units with quercetin concentrations as low as 10 M! For easy comparison, these results

176 have been plotted together in Figure 3.

177 As quercetin is not a compound extensively represented in every class of antioxidant-rich

178 foods, (+)-catechin was also analyzed using the traditional CAA assay in HepG2 cells (Figure 4).

179 In contrast to that observed with quercetin, the CAA of catechin was surprisingly rather weak.

180 While 250 M quercetin reduced fluorescence considerably for a CAA value of 58.61.0, the

181 same concentration of catechin resulted in a CAA value of only 16.50.9. Wolfe and Liu

182 examined the structureactivity relationships of pure compounds and their effectiveness in

183 HepG2-based CAA measurements and found that catechin had virtually no antioxidant activity,
184 while quercetin was extremely active (Wolfe & Liu, 2008). Structural characteristics of catechin,

185 such as the ortho-dihydroxy moiety on the B-ring and the C-3 hydroxy group, both increase

186 catechins radical-scavenging/antioxidant potential relative to that of quercetin (Bors, Heller,

187 Michel, & Saran, 1990). As monomeric and polymeric catechin/epicatechin and their derivatives

188 are prevalent in tree nuts, it was determined that the HepG2-based CAA assay would not be

189 useful for gaining biologically relevant information on the antioxidant potential of tree nuts such

190 as U.S. pecans. For this reason further experiments were performed using the Caco-2 cell line.

191 Results for both (+)-catechin and quercetin were quite different in the Caco-2 cell line. As

192 evident in Figure 5, CAA values for catechin were superior in Caco-2 cells: catechin imparted a

193 greater reduction in fluorescence than previously observed in HepG2 cells. Overall in Caco-2

194 cells, fluorescence was reduced by 25.5 to 61.0% across the varying concentrations of catechin

195 tested; whereas in HepG2 cells, the maximum reduction in fluorescence was much less, at only

196 16.5%. Rodriguez-Mateos et al. (2014) reported that the cellular accumulation of ()-epicatechin

197 after a 1 h incubation was more pronounced in Caco-2 monolayer cells (239 pmol/mg protein)

198 than HepG2 cells (56 pmol/mg protein).

199 The cellular effects of flavonoids will ultimately depend on the extent to which they associate

200 with the cell type in question, either by interactions with membrane or uptake into the cytosol

201 (Rodriguez-Mateos et al., 2014). One possible explanation for the difference in the CAA

202 between the two cell lines is their membrane transport systems (Lodish et al., 2003). As passive

203 diffusion through membranes is similar for all cells, differences in active membrane transport is

204 likely responsible for the observed differences in the CAA. Unfortunately, the role of

205 transporters in both cellular accumulation and cellular efflux has only recently been appreciated

206 and our knowledge for individual phenolic compounds is at a basic level.
207 We have studied the antioxidant activity of phenolic extracts from pecans in Caco-2 cells:

208 they behaved similarly to catechin at a 30 M concentration (unpublished data). As there have

209 been no previous CAA studies using phenolics derived from tree nuts, direct comparison to the

210 literature is not possible. Previous research performed by Wan et al. (2015) aimed to optimize

211 the CAA assay for Caco-2 cells. However, Wan et al. (2015) did not examine the same

212 compounds in multiple cell lines. These authors concluded that Caco-2 cells could be used very

213 effectively for such measurements of biological antioxidant activity, which has been further

214 demonstrated here with food antioxidants.

215

216 3.2. Cytotoxicity results

217 For the MTT viability assay, quercetin and (+)-catechin standards were applied to both cell

218 lines at concentrations ranging between 2.5 and 250 M for 1 h; no statistically significant

219 effects (p > 0.05) between the sample and control cells were observed. From this, we can

220 conclude that the phenolics were not toxic to the cells at any of the concentrations examined in

221 the study and that reduced fluorescence was resultant from antioxidant defenses.

222

223 4. Conclusions

224 In summation, CAA results from Caco-2 cells (known to be good models of the intestinal

225 barrier) were directly compared to those from HepG2 cells. Caco-2 cells yielded more

226 antioxidant activity (shown as substantial reductions in fluorescence) for two common food

227 phytochemicals, as well as being able to be a potentially suitable cell line to determine the

228 antioxidant activity of tree nut extracts. As HepG2 cells were not suitable for flavan-3-ols, such
229 as catechin, the Caco-2 cell model would be more appropriate for assessing foods rich in these

230 phenolics.

231

232 Acknowledgments

233 The authors would like to acknowledge the USDA-NIFA-SCRI Award No. 2011-51181-30674

234 and the Georgia Agricultural Commodity Commission for Pecans (GACCP) for funding this

235 research.

236

237 Conflict of interest

238 The authors have no conflict of interest.

239

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Figure captions

Figure 1. Possible mechanisms of action of antioxidants in cells incubated with 2,7-

dichlorofluorescin diacetate (DCFH-DA). They may (1) competitively quench free

radicals before they can reach the 2,7-dichlorofluorescin (DCFH) dye; (2) react with

the 2,2-azobis (2-amidinopropane) dihydrochloride (ABAP) directly to inhibit the

formation of radicals; (3) inhibit lipid peroxidation; (4) react with alkyl peroxy

radicals to stop propagation of other radicals; and (5) inhibit intracellular redox

pathways that can also oxidize the DCFH (Wolfe & Liu, 2007; Wardman, 2008;

Lpez-Alarcn, & Denicola, 2013). Additional abbreviations are as follows: ROO,

alkyl peroxy radical; ROS/RNS, reactive oxygen species/reactive nitrogen species;

XH/X, antioxidant/antioxidant radical; LH/LOO, lipid/lipid peroxy radical; and

DCF, 2,7-dichlorofluorescein.

Figure 2. Chemistry of the 2,7-dichlorofluorescin diacetate (DCFH-DA) after it enters the

HepG2 or Caco-2 cell lines.

Figure 3. Cellular antioxidant activity (CAA) of quercetin (meanstandard error based on

triplicate determinations of four separate experiments) in HepG2 cells. Cells,

incubated in 96-well culture plates, were treated with equal volumes of 25 M

DCFH-DA and various concentrations of quercetin for 1 h and then washed and

incubated with 600 M ABAP. Fluorescence was measured for 1 h at the

excitation/emission wavelength pair of 485 nm and 538 nm; CAA units represent
 cellular antioxidant capacity. Error bars not visible signify values < the symbol size

used. Our results ( ) are compared against those of Wolfe and Liu ( ).10

Figure 4. Cellular antioxidant activity (CAA) of quercetin ( ) and (+)-catechin ( ) in

HepG2 cells. CAA units, a value reflecting cellular antioxidant capacity, were

determined at the fluorescence excitation/emission wavelength pair of 485 nm and

538 nm. The data represent meanstandard error based on triplicate determinations of

four separate experiments. Error bars not visible signify values < the symbol size

used.

Figure 5. Cellular antioxidant activity (CAA) of quercetin (meanstandard error based on

triplicate determinations of four separate experiments) using HepG2 ( ) and Caco-2

( ) cell types, as well as the CAA of (+)-catechin (meanstandard error based on

triplicate determinations of four separate experiments) using HepG2 ( ) and Caco-2

( ) cell types. Error bars not visible signify values < the symbol size used.
Figure 1.
Figure 2.
 60
CAA Unit

40

20

0
0 50 100 150 200 250

Quercetin (M)

Figure 3.
 60

50

40
CAA Unit

30

20

10

0
0 50 100 150 200 250
Phenolic concentration (M)

Figure 4.
 70

60

50
CAA Unit

40

30

20

10

0
0 50 100 150 200 250
(+)-Catechin/Quercetin Concentration (M)

Figure 5.
Highlights

Choosing an appropriate cell line to evaluate the potential of food antioxidants is crucial.

Most cellular antioxidant activity (CAA) assays of foods use a HepG2 cell model, which is

not ideal.

A Caco-2 cell line was found to be superior for the CAA assay when analyzing flavan-3-ols.

Comparing the CAA of quercetin and catechin with both cells revealed Caco-2 to be more

utilitarian.