Identification of Black Bean (Phaseolus vulgaris L.) Polyphenols That

Jun 5, 2015 - Journal of Agricultural and Food Chemistry. Hu, Cheng, Heller, Krasnoff, Glahn and Welch. 2006 54 (24), pp 9254–9261. Abstract: Four d...
2 downloads 16 Views 710KB Size
Article pubs.acs.org/JAFC

Identification of Black Bean (Phaseolus vulgaris L.) Polyphenols That Inhibit and Promote Iron Uptake by Caco‑2 Cells Jonathan J. Hart,* Elad Tako, Leon V. Kochian, and Raymond P. Glahn Robert W. Holley Center for Agriculture and Health, Agricultural Research Service, U.S. Department of Agriculture, Cornell University, Ithaca, New York 14853, United States ABSTRACT: In nutritional studies, polyphenolic compounds are considered to be inhibitors of Fe bioavailability. Because they are presumed to act in a similar manner, total polyphenols are commonly measured via the Folin−Ciocalteu colorimetric assay. This study measured the content of polyphenolic compounds in white and black beans and examined the effect of individual polyphenols on iron uptake by Caco-2 cells. Analysis of seed coat extracts by LC-MS revealed the presence of a range of polyphenols in black bean, but no detectable polyphenols in white bean. Extracts from black bean seed coats strongly inhibited iron uptake. Examination of the eight most abundant black bean seed coat, non-anthocyanin polyphenols via Caco-2 cell assays showed that four (catechin, 3,4-dihydroxybenzoic acid, kaempferol, and kaempferol 3-glucoside) clearly promoted iron uptake and four (myricetin, myricetin 3-glucoside, quercetin, and quercetin 3-glucoside) inhibited iron uptake. The four inhibitors were present in 3-fold higher total concentration than the promoters (143 ± 7.2 vs 43.6 ± 4.4 μM), consistent with the net inhibitory effect observed for black bean seed coats. The ability of some polyphenols to promote iron uptake and the identification of specific polyphenols that inhibit Fe uptake suggest a potential for breeding bean lines with improved iron nutritional qualities. KEYWORDS: polyphenols, iron bioavailability, Caco-2 cells, catechin, myricetin



INTRODUCTION Iron-deficiency anemia is the most common micronutrient deficiency in the world. The World Health Organization estimates that nearly 25% of the planet’s population is iron (Fe) deficient.1 The problem is most pressing among poor populations that derive iron nutrition primarily from nondiverse plant-based diets. Many staple plant food crops contain phytochemical components that inhibit mineral micronutrient bioavailability, typically by binding and preventing mineral uptake by intestinal cells. Two classes of such compounds are inositol phosphates (e.g., phytic acid) and polyphenols. Inositol hexakisphosphate (IP6) is the predominant phytate form in most unprocessed seeds and is almost always in high molar excess relative to Fe, commonly exhibiting molar ratios of 8:1 (phytate/Fe) or higher in legumes.2 Like other organic acids such as citrate, phytate is known to bind Fe and, although the Fe−phytate complex can remain soluble, high molar excess prevents interaction and exchange of free Fe with the intestinal brush border surface and Fe transporters and therefore limits bioavailability. Phytic acid does not reduce Fe and thus complexes with Fe in the ferric state.3 Polyphenols are present in a wide variety of foods, including staple crops such as wheat,4 potato,5 soybean,6 sorghum,7 and common bean.8 Polyphenols are known inhibitors of Fe bioavailability and are presumed to act in a manner similar to phytate by complexing Fe. Many studies have demonstrated a high binding capacity of polyphenols with Fe.9−12 Interestingly, some polyphenols have also been shown to be capable of reducing Fe(III) to Fe(II),13 and this presents the possibility that they may have the capacity to promote Fe bioavailability. Although a substantial amount of information is known about binding of Fe by polyphenols, little has been done to characterize Fe-uptake inhibition of specific polyphenols and This article not subject to U.S. Copyright. Published XXXX by the American Chemical Society

to identify polyphenols that may actually be able to promote Fe absorption. Thus, increased knowledge of the presence and effects of specific polyphenols represents a potential avenue to enhanced bioavailability of Fe in staple food crops. Such analysis is very much relevant to a number of international breeding programs that are aimed at alleviating dietary mineral deficiencies by increasing Fe content in staple food crops such as various beans,14−17 rice,18 and pearl millet.19 Nutritional Fe bioavailability research can be undertaken via a range of study models. Human feeding trials are perhaps the most direct way of examining the effects of food components on Fe nutrition and have been employed in studies of Fe absorption from common beans.20,21 Petry et al.22 reported that polyphenols and phytate in common beans can contribute to decreased Fe absorption in young women. Another approach to studying nutrient availability is through the use of animal models. Although animal models only approximate human nutritional physiology, they allow good dietary control in studies of the nutritional effects of particular food components. A recent study with Fe-deficient broiler chickens showed that iron-fortified black beans produced limited Fe dietary benefit (i.e., improved Fe status), likely due to the presence of high levels of polyphenols in black bean seed coats.17 Similarly, higher iron bioavailability from white beans compared with red beans was demonstrated in a feeding trial with week-old chicks, again implicating the role of higher polyphenolic content in red beans for the decrease in iron nutrition.15 Received: February 3, 2015 Revised: May 14, 2015 Accepted: June 5, 2015

A

DOI: 10.1021/acs.jafc.5b00531 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

solutions were further diluted with pH 2 medium to appropriate concentrations for use in Caco-2 assays. Maximum DMSO concentration in the highest polyphenol concentration treatments (30 μM) was 3.1%. For use as LC-MS standards, polyphenols were dissolved to a concentration of 4 mM in DMSO and diluted to 1 mM with MeOH/H2O (50:50 v/v). These stock solutions were further diluted with 50:50 MeOH/H2O for use in standard curves. Caco-2 Assays. Assays were performed with Caco-2 cells cultured on the bottom surface of wells in 24-well culture plates (Costar 24 Well Clear TC-Treated Multiple Well Plates). Cells were grown in DMEM supplemented with 3.7 g/L sodium bicarbonate, 25 mM HEPES (pH 7.2), and 10% fetal bovine serum. Cells were seeded at passages 29−38, at a density of 50000 cells/cm2 on collagen-treated plates. Under these culture conditions, the cells were confluent by day 4 postseeding and formed highly reproducible, differentiated cell monolayers with consistent protein concentrations over the course of numerous experiments. Cells were used in experiments 13 days postseeding. Solutions containing bean extract and/or polyphenol(s) were prepared in 150 μL of pH 2 medium. Fifty microliters of iron standard diluted in the same medium was added to extract or polyphenol solutions to achieve 4 or 10 μM Fe final concentration, and combined solutions (200 μL) were incubated at room temperature for 15 min before addition of 1 mL of MEM, supplemented with 3.5 g/L glucose, 2.2 g/L sodium bicarbonate, 10 mmol/L piperazine-N,N′-bis[2-ethanesulfonic acid] (PIPES) (pH 7.0), 1% antibiotic−antimycotic solution, hydrocortisone (4 mg/L), insulin (5 mg/L), selenium (5 μg/L), triiodothyronine (34 μg/L), and epidermal growth factor (20 μg/L). A half milliliter of this combined solution was applied directly onto Caco-2 cell monolayers in 24-well plates. After overnight incubation at 37 °C, cells were washed twice with a buffered saline solution (130 mM NaCl, 5 mM KCl, 5 mM PIPES, pH 6.7) and then lysed by the addition of 500 μL of 18 MΩ water. Lysed cells were measured for ferritin and protein concentrations as described previously24,25 with modifications. Ferritin data in Figures 1−4 were acquired using an immunoradiometric assay (FER-IRON II), and data in Figure 5 used an enzyme immunoassay (SPECTRO FERRITIN, both from RAMCO Laboratories, Stafford, TX, USA). Ferritin formation was used as a measure of iron uptake and was expressed in nanograms of ferritin per milligram of cell protein. Cell protein was quantified using the Bio-Rad DC protein assay kit (Bio-Rad Laboratories). Means and standard errors of three replications were calculated for both protein and ferritin concentrations. LC-MS Analysis. Extracts and standards were analyzed with a Waters Acquity UPLC. Five microliter samples were injected and passed through an Acquity UPLC BEH Shield RP18, 1.7 μm, 2.1 × 100 mm, column (Waters) at 0.5 mL/min. The column was temperature-controlled at 40 °C. The mobile phase consisted of water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B). Polyphenols were eluted using linear gradients of 86.7−84.4% A in 1.5 min, 84.4−81.5% A in 0.2 min, 81.5−77% A in 2.8 min, 77−55% A in 0.5 min, 55−46% A in 1 min, and 46−86.7% A in 0.2 min and a 0.8 min hold at 86.7% A for a total 7 min run time. From the column, flow was directed into a Waters Acquity photodiode array detector set at 300−400 nm and a sampling rate of 20/s. Flow was then directed into the source of a Xevo G2 QTOF mass spectrometer (Waters Corp.), and ESI mass spectrometry was performed in negative ionization mode with a scan speed of 5/s in the mass range from 50 to 1200 Da. Capillary and cone gas voltages were set at 2.3 kV and 30 V, respectively. Desolvation gas flow was 800 L/h, and desolvation gas temperature was 400 °C. Source temperature was 140 °C. Lock-mass correction was used, with leucine enkephalin as the lock-mass standard and a scan frequency of 25 s. Instrumentation and data acquisition were controlled by MassLynx software. Individual polyphenols in bean samples were tentatively determined by mass using MarkerLynx software, and their identities were confirmed by comparison of LC retention times with authentic standards. Polyphenol standard curves for flavonoids were derived from integrated areas under UV absorption peaks from 10 replications.

In vitro models using cultured human intestinal cells have been shown to be useful for a more focused investigation of the absorption of dietary compounds. One such model, Caco-2 cultured intestinal cells, has been successfully used to study the characteristics of transepithelial transport of a variety of nutrients and dietary components.23 This system has been adapted for use in iron uptake research24 and has been used extensively to study iron nutritional qualities of a range of foods and food products. An advantage of the Caco-2 system is that it confers the ability to study Fe uptake effects of individual food components apart from other dietary factors. Using this in vitro system, Hu et al.25 reported that flavonoid polyphenols in colored bean seed coats strongly inhibited Fe bioavailability in bean digests. Thus, polyphenols have been implicated in reduced iron nutrition, specifically in beans, by studies using a range of experimental systems. In Fe bioavailability studies, polyphenols have commonly been grouped as Fe uptake inhibitors, with the implication that all members of this class of phytochemicals act in a similar manner. The generalized nature of the presumed inhibitory effect is illustrated by the way that polyphenols are typically measured. Most studies use the Folin−Ciocalteu method,26 and results are reported as gallic acid equivalents.27 Little information has been established concerning the effects of specific individual polyphenols on Fe bioavailability. This work set out to identify polyphenolic compounds present in black and white bean and to examine the relative Fe-inhibition properties of specific polyphenols using a Caco-2 cell assay.



MATERIALS AND METHODS

Chemicals. LC-MS grade acetonitrile, LC-MS grade methanol, LCMS grade formic acid, quercetin 3-glucoside, kaempferol 3-glucoside, 3,4-dihydroxybenzoic acid, catechin, myricetin, quercetin, kaempferol, dimethyl sulfoxide (DMSO), glucose, hydrocortisone, insulin, selenium, triiodothyronine, and epidermal growth factor were purchased from Sigma-Aldrich (St. Louis, MO, USA). Myricetin 3glucoside was purchased from Extrasynthese (Genay, France). Sodium bicarbonate and PIPES were from Fisher Scientific (Waltham, MA, USA). Fe standard (1000 μg/mL in 2% HCl) was from High-Purity Standards (Charleston, SC, USA). Modified Eagle’s medium (MEM), Dulbecco’s modified Eagle’s medium (DMEM), and 1% antibiotic− antimycotic solution were purchased from Gibco (Grand Island, NY, USA). Bean Sample Preparation. Dried black and “Great Northern” white beans (Goya Foods, Inc., Secaucus, NJ, USA) were purchased at a local market. Isolated seed coats were prepared by wrapping whole black and white beans in deionized water-soaked paper towels until seed coats began to wrinkle and separate from cotyledons. Seed coats were then removed with forceps, dried, and ground to a coarse powder with a mortar and pestle. Whole beans were rinsed with deionized water, autoclaved for 30 min, lyophilized, and ground with a coffee grinder. To 1 g of ground bean material (cooked whole beans or isolated seed coats) was added 5 mL of methanol/water (50:50 v/v). The slurry was vortexed for 1 min, placed in a 24 °C sonication water bath for 15 min, vortexed again for 1 min, and centrifuged at 4000g for 15 min. The supernatant was filtered with a 0.45 μm Teflon syringe filter and stored for later use in a −20 °C freezer. For use in Caco-2 assays, whole bean methanol/water extracts were dried in a rotary evaporator, weighed, and redissolved in an appropriate volume of pH 2 medium (140 mM NaCl and 5 mM KCl, pH adjusted with HCl) to achieve the desired extract concentration. Preparation of Fe and Polyphenol Solutions. Fe stock solutions were prepared from 1000 mg/mL Fe standard in pH 2 medium. For use in Caco-2 assays, polyphenols were dissolved to a concentration of 1.6 mM in DMSO and diluted with pH 2 medium to achieve 400 μM stock solutions containing 25% DMSO. Stock B

DOI: 10.1021/acs.jafc.5b00531 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

Standard curves for catechin and 3,4-dihydroxybenzoic acid were constructed from MS ion intensities using 10 replications. Statistical Analysis. Data are presented as the mean ± standard error of the mean (SEM) in Caco-2 assays of nanograms of ferritin per milligram of protein (Figures 1−5) and as the mean ± standard deviation (SD) for polyphenol concentrations (Table 1).

Table 1. Concentrations of Polyphenols Present in Extracts from Black Bean Seed Coatsa polyphenol

rt (min)

m/z

myricetin 3-O-glucoside quercetin 3-O-glucoside kaempferol 3-O-glucoside 3,4-dihydroxybenzoic acid catechin myricetin quercetin kaempferol

2.80 3.69 4.54 1.03 1.35 5.39 5.80 6.26

479.0820 463.0870 447.0919 153.0179 289.0706 317.0292 301.0344 285.0399

mean ± SD (μM)

p/i

± ± ± ± ± ± ± ±

i i p p p i i p

100.6 33.2 17.6 14.3 10.2 7.4 1.8 1.5

5.1 1.6 0.9 2.5b 0.9b 0.4 0.1 0.1

Figure 2. Ferritin formation in Caco-2 cells in response to various concentrations of extracts from white and black bean seed coats. Dotted line indicates ferritin response to 10 μM Fe alone. Error bars represent standard error of the mean and do not extend outside some data points.

a

rt, retention time; p/i, promoter or inhibitor of iron uptake in Caco-2 assays. bQuantified by MS ion intensity. Other concentrations were determined by integration of UV absorption peak areas.



of ≥0.2 mg/mL, ferritin production was double that of 10 μM Fe applied alone. Baseline levels of ferritin production were observed in the presence of both white and black bean seed coat extracts when Fe was withheld, demonstrating that enhanced ferritin production by white bean seed coats was not associated with higher endogenous Fe concentration in white bean seed coats. In striking contrast to the response to white bean seed coat extracts, black bean seed coat extracts strongly inhibited ferritin formation to nearly cell baseline levels at extract concentrations of ≥0.2 mg/mL (Figure 2). Because it has been reported that polyphenols inhibit iron absorption in Caco-2 cells,25,28 we investigated the effects of individual polyphenols on ferritin formation. Various concentrations of the flavonol myricetin, a polyphenol known to be present in black beans,8 were added to whole black and white bean extracts (0.2 mg/mL) in a Caco-2 ferritin assay to test the response. Ferritin formation was inhibited in both black and white beans in the presence of myricetin at concentrations ≥10 μM Fe (Figure 3). In the absence of bean extract, 20 μM myricetin plus 10 μM inhibited ferritin formation down to cell baseline levels. When supplied alone to Caco-2 cells, specific polyphenols had markedly varying effects on ferritin formation. At concentrations equimolar and greater than that of Fe, myricetin strongly inhibited ferritin formation (Figure 4). At higher myricetin concentrations, ferritin formation declined to cell baseline levels. The flavanol epicatechin induced a strong promotional effect on ferritin formation at all concentrations (Figure 4). The increase in ferritin production in the presence of epicatechin was Fe-dependent, as indicated by the low (cell baseline) level of ferritin formed when cells were supplied with 20 μM epicatechin in the absence of Fe (Figure 4). To identify specific polyphenol composition, black and white bean seed coat extracts were analyzed by LC-MS. In black bean, the most abundant identifiable polyphenol was myricetin 3-Oglucoside, followed by lesser amounts of quercetin 3-Oglucoside and kaempferol 3-O-glucoside (Table 1). The benzoic acid, 3,4-dihydroxybenzoic acid, and the flavanol catechin were also present, followed by aglycones of the most abundant glycosylated flavonols (Table 1). White bean seed coat extracts contained no detectable amounts of these

RESULTS Increasing concentrations of extracts from whole white or black beans applied to Caco-2 cells in the presence of 10 μM Fe induced an increase in ferritin formation, reaching a maximum at extract concentrations of 0.1−0.2 mg/mL (Figure 1). At

Figure 1. Ferritin formation in Caco-2 cells in response to various concentrations of extracts from whole white and black beans. Dotted line indicates ferritin response to 10 μM Fe alone. Error bars represent standard error of the mean and do not extend outside some data points.

higher extract concentrations, ferritin formation declined, with a greater decrease observed in black bean extracts. In the absence of added Fe, ferritin formation remained around baseline levels in both white bean and black bean. When extracts from seed coats isolated from both black and white beans were applied to Caco-2 cells, the ferritin formation responses to the two types of bean were very different. The presence of white bean seed coats promoted ferritin formation at all extract concentrations, whereas seed coat extracts from black bean inhibited ferritin formation at all concentrations (Figure 2). In white bean seed coats, at extract concentrations C

DOI: 10.1021/acs.jafc.5b00531 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

polyphenols were repeated several times to confirm those observations.



DISCUSSION In both in vitro and in vivo studies, whole colored beans and the seed coats from colored beans have consistently been shown to inhibit Fe bioavailability, and this inhibition has been demonstrated to be due to the presence of polyphenols in the seed coat.2,15,22,29 As expected, in the present study, extracts of black bean whole seeds or seed coats inhibited ferritin production (i.e., cell Fe uptake) in Caco-2 cells to a much greater degree than similar extracts from white bean (Figures 1 and 2). The results clearly show that when the black bean seed coat is present, either as an extract from seed coats or whole seeds, the relative effect is one of inhibition of Fe uptake. It should be noted that, although the primary focus of this study was the effects of polyphenols on Fe uptake, extracts from whole black and white beans and seed coats of white bean produced an enhancement of Fe uptake compared with supplying Caco-2 cells with Fe alone (Figures 1 and 2). The factor(s) present in cotyledons or seed coats responsible for increased ferritin formation cannot be determined from these results, but we can speculate that proteins and possibly phytate and/or other compounds present in extracts could help to solubilize Fe and make it available for absorption. This effect was concentration-dependent, with a maximum Fe uptake at about 0.2 mg/mL in both whole white and black bean extracts (Figure 1). This effect may be due, at least in part, to the presence of phytic acid in cotyledons. Under some conditions, low levels of phytic acid can actually promote Fe uptake in vitro,30 whereas higher levels like those normally found in crops such as beans inhibit Fe uptake. As expected, the addition of ascorbic acid to Fe in controls (Figures 1−5) clearly demonstrated the power of this reducing agent to increase Fe uptake in Caco-2 assays. It is also important to note that the low (near cell baseline) level of ferritin production in the absence of added Fe indicates that the compounds extracted from whole beans or seed coats (Figures 1 and 2) or individual polyphenols applied alone to Caco-2 cells (Figures 4 and 5) did not by themselves cause the ferritin formation response. Also, given that the extracts contained negligible amounts of Fe, it is very likely that the ferritin formation responses observed in these experiments were due to the interaction of extracted compounds with exogenously added Fe. Therefore, we can be confident that the in vitro conditions used in this work are suitable for characterizing both promotional and inhibitory effects of extractable compounds on Caco-2 cell Fe uptake. There has been a consistent general assertion in the literature that all polyphenols inhibit Fe bioavailability.31 This assumption is countered, however, by the results of the present study. Clearly, the addition of the flavonoid myricetin, a polyphenol known to be present in black beans,8 to whole black and white bean extracts brought about a concentration-dependent decrease in Fe bioavailability (Figure 3). At myricetin concentrations >5 μM, the ferritin-promoting effects of whole bean extracts seen in Figure 1 were reversed by the addition of this individual polyphenol. Similarly, direct application of myricetin alone to Caco-2 cells brought about a strong decrease in Fe uptake at concentrations >5 μM (Figure 4). On the other hand, the strong, concentration-dependent promotion of ferritin formation by epicatechin and catechin (Figures 4 and 5) and the moderate enhancement of Fe uptake

Figure 3. Ferritin formation in Caco-2 cells in response to various concentrations of myricetin plus 0.2 mg/mL extracts from whole white and black beans. Dotted line indicates ferritin response to 10 μM Fe alone. Error bars represent standard error of the mean and do not extend outside some data points.

Figure 4. Ferritin formation in Caco-2 cells in response to various concentrations of epicatechin and myricetin, without bean extract. Dotted line indicates ferritin response to 10 μM Fe alone. Error bars represent standard error of the mean and do not extend outside some data points.

compounds. Anthocyanidins and anthocyanins, although present in black bean, were not considered in this study. To examine the Fe bioavailability effects of the most abundant polyphenols present in black bean seed coats, each of the eight compounds listed in Table 1 was tested for its effect on ferritin formation with the Caco-2 assay. Kaempferol 3-Oglucoside, kaempferol, catechin, and 3,4-dihydroxybenzoic acid promoted ferritin production, whereas myricetin 3-O-glucoside, quercetin 3-O-glucoside, quercetin, and myricetin inhibited the formation of ferritin in a concentration-dependent manner (Figure 5). For all eight compounds, withholding Fe resulted in cell baseline levels of ferritin production. Relative Caco-2 ferritin responses varied somewhat among experiments as shown by slightly different values of Fe-alone and Fe plus ascorbic acid treatments. This is normal for Caco-2 bioassays, and it is important to note that in all cases, polyphenols showing either promotional or inhibitory effect on Fe bioavailability did so consistently. Experiments with individual D

DOI: 10.1021/acs.jafc.5b00531 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

Figure 5. Ferritin formation in Caco-2 cells in response to various concentrations of eight polyphenols determined to be present in black bean seed coat extracts. Dotted line indicates ferritin response to 4 μM Fe alone. Error bars represent standard error of the mean and do not extend outside some data points.

pH. Catechol32 and epicatechin33,34 have been demonstrated to reduce Fe3+ to Fe2+ at pH 2−3. In our experiments, polyphenols were incubated with Fe in pH 2 solution for 15 min and then added to pH 7-buffered MEM and quickly placed on Caco-2 cell monolayers. It is conceivable that this protocol was capable of generating sufficient Fe2+ to cause promotion of iron uptake before re-oxidation of Fe2+ to Fe3+ occurred. Reoxidation of Fe2+ has been reported to occur with an increase in pH, but the kinetics with which this may occur is not well characterized.32 Iron reduction via the catecholate configuration on the B ring of epicatechin and catechin may be involved with increased Fe uptake.13 Another promoter of iron uptake in our study, 3,4dihydroxybenzoic acid, also contains a catecholate substructure. However, the promoters kaempferol and kaempferol 3glucoside do not have either catecholate or gallate substructures, so their Fe-uptake promotional properties may proceed through another mechanism. On the other hand, the inhibitors quercetin, myricetin, and their glucosides do contain catecholate and gallate ligands, respectively. The inhibitory properties of those four polyphenols may occur simply by strongly binding iron and making it unavailable for uptake. Thus, a consistent polyphenol structure/iron bioavailability functional relationship influencing Fe bioavailability is not apparent in the major black bean polyphenols identified in this study. We can speculate on the concentration-dependent inhibitory activity of myricetin on iron uptake (Figure 4) on the basis of

by kaempferol, kaempferol 3-glucoside, and 3,4-dihydroxybenzoic acid (Figure 5), all applied directly to Caco-2 cells, are in contrast to the general assumption that polyphenols inhibit Fe uptake. As summarized in Table 1, of the eight most abundant non-anthocyanin polyphenols present in black bean seed coats, four were shown to be promoters of iron uptake in Caco-2 cells and four were shown to be inhibitors (Figure 5). This appears to be the first study to clearly show both inhibitory and promotional effects on Fe uptake by specific polyphenols. It is important to note that these cell culture experiments are relatively easy to carry out. Thus, we believe that this approach can be applied to characterize the effects of the multitude of other compounds present in staple food crops (and their interactions) that can influence Fe bioavailability. The mechanism by which epicatechin and catechin promote Fe uptake is not known. One possible mechanism that may be involved with promotion is increased solubility of iron− epicatechin/catechin complexes coupled with favorable exchange kinetics at membrane-bound iron transport sites. This would tend to increase the amount of iron available for uptake by Caco-2 cells. Another possible mechanism with some support in the literature is reduction of Fe3+ to Fe2+ by epicatechin. As shown in Figures 1−5, iron reduction by ascorbic acid had a strongly promoting effect on iron uptake in our Caco-2 system. If it occurs, iron reduction by epicatechin should also have a promoting effect. As reviewed by Perron et al.,13 reduction of Fe3+ to Fe2+ by some polyphenols can occur, especially at low E

DOI: 10.1021/acs.jafc.5b00531 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

(3) Torres, J.; Dominguez, S.; Cerda, M. F.; Obal, G.; Mederos, A.; Irvine, R. F.; Diaz, A.; Kremer, C. Solution behavior of myo-inositol hexakisphosphate in the presence of multivalent cations. Prediction of a neutral pentamagnesium species under cytosolic/nuclear conditions. J. Inorg. Biochem. 2005, 99, 828−840. (4) Asenstorfer, R. E.; Wang, Y.; Mares, D. J. Chemical structure of flavonoid compounds in wheat (Triticum aestivum L.) flour that contribute to the yellow colour of Asian alkaline noodles. J. Cereal Sci. 2006, 43, 108−119. (5) Dao, L.; Friedman, M. Chlorogenic acid content of fresh and processed potatoes determined by ultraviolet spectrophotometry. J. Agric. Food Chem. 1992, 40, 2152−2156. (6) Franke, A. A.; Hankin, J. H.; Yu, M. C.; Maskarinec, G.; Low, S. H.; Custer, L. J. Isoflavone levels in soy foods consumed by multiethnic populations in Singapore and Hawaii. J. Agric. Food Chem. 1999, 47, 977−986. (7) Beta, T.; Rooney, L. W.; Marovatsanga, L. T.; Taylor, J. R. N. Phenolic compounds and kernel characteristics of Zimbabwean sorghums. J. Sci. Food Agric. 1999, 79, 1003−1010. (8) Lin, L.-Z.; Harnly, J. M.; Pastor-Corrales, M. S.; Luthria, D. L. The polyphenolic profiles of common bean (Phaseolus vulgaris L.). Food Chem. 2008, 107, 399−410. (9) Sungur, S.; Uzar, A. Investigation of complexes tannic acid and myricetin with Fe(III). Spectrochim. Acta, Part A 2008, 69, 225−229. (10) Mladenka, P.; Macakova, K.; Filipsky, T.; Zatloukalova, L.; Jahodar, L.; Bovicelli, P.; Silvestri, I. P.; Hrdina, R.; Saso, L. In vitro analysis of iron chelating activity of flavonoids. J. Inorg. Biochem. 2011, 105, 693−701. (11) Hynes, M. J.; Coinceanainn, M. O. The kinetics and mechanisms of the reaction of iron(III) with gallic acid, gallic acid methyl ester and catechin. J. Inorg. Biochem. 2001, 85, 131−142. (12) Ryan, P.; Hynes, M. J. The kinetics and mechanisms of complex formation and antioxidant behaviour of the polyphenols EGCg and ECG with iron(III). J. Inorg. Biochem. 2007, 101, 585−593. (13) Perron, N. R.; Brumaghim, J. L. A review of the antioxidant mechanisms of polyphenol compounds related to iron binding. Cell Biochem. Biophys. 2009, 53, 75−100. (14) Tako, E.; Glahn, R. P.; Laparra, J. M.; Welch, R. M.; Lei, X.; Kelly, J. D.; Rutzke, M. A.; Miller, D. D. Iron and zinc bioavailabilities to pigs from red and white beans (Phaseolus vulgaris L.) are similar. J. Agric. Food Chem. 2009, 57, 3134−3140. (15) Tako, E.; Glahn, R. P. White beans provide more bioavailable iron than red beans: studies in poultry (Gallus gallus) and an in vitro digestion/Caco-2 model. Int. J. Vitam. Nutr. Res. 2010, 80, 416−429. (16) Tako, E.; Blair, M. W.; Glahn, R. P. Biofortified red mottled beans (Phaseolus vulgaris L.) in a maize and bean diet provide more bioavailable iron than standard red mottled beans: studies in poultry (Gallus gallus) and an in vitro digestion/Caco-2 model. Nutr. J. 2011, 10, 113−122. (17) Tako, E.; Beebe, S. E.; Reed, S.; Hart, J. J.; Glahn, R. P. Polyphenolic compounds appear to limit the nutritional benefit of biofortified higher iron black bean (Phaseolus vulgaris L.). Nutr. J. 2014, 13, 28. (18) Grieger, J. A.; Haas, J. D.; Murray-Kolb, L. E.; Kris-Etherton, P.; Beard, J. L. Nutrient adequacy and food group consumption of Filipino novices and religious sisters over a nine month period. Asia Pac. J. Clin. Nutr. 2008, 17, 566−572. (19) Tako, E.; Reed, S. M.; Budiman, J.; Hart, J. J.; Glahn, R. P. Higher iron pearl millet (Pennisetum glaucum L.) provides more absorbable iron that is limited by increased polyphenolic content. Nutr. J. 2015, 14, 11. (20) Lynch, S. R.; Beard, J. L.; Dassenko, S. A.; Cook, J. D. Iron absorption from legumes in humans. Am. J. Clin. Nutr. 1984, 40, 42− 47. (21) Donangelo, C. M.; Woodhouse, L. R.; King, S. M.; Toffolo, G.; Shames, D. M.; Viteri, F. E.; Cheng, Z.; Welch, R. M.; King, J. C. Iron and zinc absorption from two bean (Phaseolus vulgaris L.) genotypes in young women. J. Agric. Food Chem. 2003, 51, 5137−5143.

the well-documented ability of phenolic compounds to chelate iron.35−37 Stability constant values for Fe3+−myricetin complexes in the range of 105−109 have been reported,9 and inhibition of iron uptake due to binding by polyphenols has been demonstrated in Caco-2 cells.28,38 Thus, the iron uptake inhibition by myricetin shown in Figure 4 is consistent with previously published reports and suggests that it is due to iron− myricetin complexation and subsequent Fe uptake inhibition. In black bean seed coats, the total concentration of the four iron uptake inhibitors was >3 times higher than the concentration of the four promoters (Table 1). Thus, it is not surprising that the net effect of black bean seed coat extracts containing all eight major polyphenols is a strong inhibition of iron uptake (Figure 2). However, implicit in this calculation is that the polyphenols studied here have a similar concentration effect on promotion or inhibition. In fact, examination of the curves in Figure 5 suggests that this assumption may not be true. For example, myricetin appears to be a more effective inhibitor than quercetin at a given concentration and catechin, a more effective promoter than kaempferol. It is not possible to deduce from the data in Figure 5 the net effect of combining promoters and inhibitors. Additional studies will be needed to establish the relative effectiveness of individual polyphenols and their interactions to promote or inhibit iron bioavailability. It would appear that identification of polyphenols that can promote iron bioavailability raises the possibility of producing breeding lines with enhanced nutritional quality. The results reported here were obtained using an in vitro cell culture system, and it is not clear that the promotional effects observed using this system would extend to in vivo systems. Experiments with animal models or human feeding trials will be necessary to test the possibility. It is interesting that a human study of the effects of several polyphenols determined that, whereas gallic acid and tannic acid inhibited iron absorption, catechin produced no such inhibition.39 This observation suggests that, even if polyphenol promotion of iron uptake in cell culture does not translate to promotion in vivo, breeding for reduced levels of inhibitory polyphenols as well as enhanced levels of promoting polyphenols may yield more nutritious staple foods.



AUTHOR INFORMATION

Corresponding Author

*(J.J.H.) Phone: (607) 254-4919. Fax: (607) 255-1132. E-mail: [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank Mary Bodis and Yongpei Chang for their invaluable assistance with Caco-2 experiments and for maintaining cell cultures. Expert technical advice concerning LC-MS was provided by Dr. Ted Thannhauser and Dr. Stuart Krasnoff.



REFERENCES

(1) WHO. Worldwide prevalence of anaemia 1993−2005. WHO Global Database on Anaemia; de Benoist, B., McLean, E., Egli, I., Cogswell, M., Eds.; World Health Organization: Geneva, Switzerland, 2008; pp 1−40. (2) Ariza-Nieto, M.; Blair, M. W.; Welch, R. M.; Glahn, R. P. Screening of iron bioavailability patterns in eight bean (Phaseolus vulgaris L.) genotypes using the Caco-2 cell in vitro model. J. Agric. Food Chem. 2007, 55, 7950−7956. F

DOI: 10.1021/acs.jafc.5b00531 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

Article

(22) Petry, N.; Egli, I.; Zeder, C.; Walczyk, T.; Hurrell, R. Polyphenols and phytic acid contribute to the low iron bioavailability from common beans in young women. J. Nutr. 2010, 140, 1977−1982. (23) Failla, M. L.; Chitchumronchokchai, C. In vitro models as tools for screening the relative bioavailabilities of provitamin A carotenoids in foods. HarvestPlus Technical Monograph 3; HarvestPlus: Washington, DC, USA, and Cali, Colombia, 2005; pp 1−33. (24) Glahn, R. P.; Lee, O. A.; Yeung, A.; Goldman, M. I.; Miller, D. D. Caco-2 cell ferritin formation predicts nonradiolabeled food iron availability in an in vitro digestion Caco-2 cell culture model. J. Nutr. 1998, 128, 1555−1561. (25) Hu, Y.; Cheng, Z.; Heller, L. I.; Krasnoff, S. B.; Glahn, R. P.; Welch, R. M. Kaempferol in red and pinto bean seed (Phaseolus vulgaris L.) coats inhibits iron bioavailability using an in vitro digestion/human Caco-2 cell model. J. Agric. Food Chem. 2006, 54, 9254−9261. (26) Folin, O.; Ciocalteu, V. On tyrosine and tryptophane determinations in proteins. J. Biol. Chem. 1927, 73, 627−650. (27) Singleton, V. L.; Orthofer, R.; Lamuela-Raventos, R. M. Analysis of total phenols and other oxidation substrates and antioxidants by means of Folin-Ciocalteu reagent. Method Enzymol. 1999, 299, 152− 178. (28) Ma, Q.; Kim, E.-Y.; Lindsay, E. A.; Han, O. Bioactive dietary polyphenols inhibit heme iron absorption in a dose-dependent manner in human intestinal Caco-2 cells. J. Food Sci. 2011, 76, H143−H150. (29) Lung’aho, M. G.; Glahn, R. P. Use of white beans instead of red beans may improve iron bioavailability from a Tanzanian complementary food mixture. Int. J. Vitam. Nutr. Res. 2010, 80, 24−31. (30) Engle-Stone, R.; Yeung, A.; Welch, R.; Glahn, R. Meat and ascorbic acid can promote Fe availability from Fe-phytate but not from Fe-tannic acid complexes. J. Agric. Food Chem. 2005, 53, 10276− 10284. (31) Mascitelli, L.; Goldstein, M. R. Inhibition of iron absorption by polyphenols as an anticancer mechanism. QJM 2011, 104, 459−461. (32) Powell, H. K. J.; Taylor, M. C. Interactions of iron(II) and iron(III) with gallic acid and its homologues: a potentiometric and spectrophotometric study. Aust. J. Chem. 1982, 35, 739−756. (33) Kennedy, J. A.; Powell, H. K. J. Polyphenol interactions with aluminum(III) and iron(III): their possible involvement in the podsolization process. Aust. J. Chem. 1985, 38, 879−888. (34) Hider, R. C.; Liu, Z. D.; Khodr, H. H. Metal chelation of polyphenols. Methods Enzymol. 2001, 335, 190−203. (35) Morel, I.; Lescoat, G.; Cillard, P.; Cillard, J. Role of flavonoids and iron chelation in antioxidant action. Methods in Enzymol. 1994, 234, 437−443. (36) Fernandez, M. T.; Mira, M. L.; Florencio, M. H.; Jennings, K. R. Iron and copper chelation by flavonoids: an electrospray mass spectrometry study. J. Inorg. Biochem. 2002, 92, 105−111. (37) Andjelkovic, M.; Van Camp, J.; De Meulenaer, B.; Depaemelaere, G.; Socaciu, C.; Verloo, M.; Verhe, R. Iron-chelation properties of phenolic acids bearing catechol and galloyl groups. Food Chem. 2006, 98, 23−31. (38) Kim, E.-Y.; Ham, S.-K.; Shigenaga, M. K.; Han, O. Bioactive dietary polyphenolic compounds reduce nonheme iron transport across human intestinal cell monolayers. J. Nutr. 2008, 138, 1647− 1651. (39) Brune, M.; Rossander, L.; Hallberg, L. Iron absorption and phenolic compounds: importance of different phenolic structures. Eur. J. Clin. Nutr. 1989, 43, 547−557.

G

DOI: 10.1021/acs.jafc.5b00531 J. Agric. Food Chem. XXXX, XXX, XXX−XXX