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Inhibition of Intestinal Cellular Glucose Uptake by Phenolics Extracted from Whole Wheat Grown at Different Locations

Inhibition of Intestinal Cellular Glucose Uptake by Phenolics Extracted from Whole Wheat Grown at... Hindawi Journal of Nutrition and Metabolism Volume 2018, Article ID 5421714, 10 pages https://doi.org/10.1155/2018/5421714 Research Article Inhibition of Intestinal Cellular Glucose Uptake by Phenolics Extracted from Whole Wheat Grown at Different Locations 1,2 1,2 3 Maryam Shamloo , Peter J. H. Jones, and Peter K. Eck Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba, Winnipeg, MB, Canada R3T 2N2 Department of Food Science, University of Manitoba, Winnipeg, MB, Canada R3T 2N2 Department of Human Nutritional Sciences, University of Manitoba, W569 Duff Roblin Building, 190 Dysart Road, Winnipeg, MB, Canada R3T 2N2 Correspondence should be addressed to Peter K. Eck; peter.eck@umanitoba.ca Received 7 November 2017; Accepted 28 December 2017; Published 18 March 2018 Academic Editor: Stan Kubow Copyright © 2018 Maryam Shamloo et al. *is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Whole grain consumption is associated with reduced risk of type 2 diabetes, and the underlying mechanism might be related to the actions of polyphenols. Dietary polyphenols contribute to low glycemic indices through inhibition of intestinal glucose transport proteins. *is study has two objectives: (1) to evaluate how the contents of phenolic acids in wheat vary by genetic background and growth condition and (2) to evaluate how these changes translate into physiologic relevance by investigating cellular glucose transporter inhibitions. Phenolic acids were extracted from wheat varieties grown at different locations over two crop years. *e degree of inhibition of glucose uptake into human Caco-2E cells was determined. Free and bound phenolic acid extracts of all wheat genotypes inhibited glucose uptake. Degree of glucose uptake inhibitions positively correlated with the contents of free and 2 2 bound phenolic acids, and the correlation coefficients were R � 0.91 and R � 0.89, respectively. Genotype and environment influenced the content of free and bound phenolic acids which linearly translated to the degree of glucose uptake inhibition in a model of intestinal absorption (P <0.05). Results of this work mechanistically support the hypothesis that dietary phenols positively influence the glycemic index and therefore the health properties of whole grain consumption. whole grain products is associated with a 20–30% reduction 1. Introduction in the risk of type 2 diabetes [5, 6]. For example, whole wheat Diabetes mellitus has become an emerging global health breads generate lower glycemic indices than white bread [7], problem. Currently, estimated 422 million adults are suf- which is in part attributed to polyphenols found in the bran fering from diabetes and that number will increase to 642 fraction of whole wheat [8]. *ese polyphenols are known to million by 2040 [1]. Type 2 diabetes is characterized by the be efficient in lowering blood glucose levels [9]; however, the body’s insensitivity to insulin and is characterized by increased mechanisms remain largely undetermined [6]. Some plant blood sugar levels [2]. In diabetic patients, the expression of bioactives inhibit intestinal glucose transporters [10, 11] intestinal glucose transporters has been reported to be 3- to causing a blunted glucose absorption [6, 7, 12]. 4-fold higher than healthy controls [3], and therefore, higher Wheat genetics and growing environments play a critical amount of glucose will be absorbed by these patients in role in influencing the accumulation of secondary plant a shorter period of time, leading to increased postprandial metabolites [13], including polyphenols. *e importance of the glycemia. *us, an effective treatment option for diabetes and genotype is demonstrated by the fact that winter wheat va- diabetes-related complications is to dampen or inhibit in- rieties contain twice the levels of total phenolic acids than testinal glucose transporters and/or glucose absorption. other genotypes [14]. *e impact of the environment is very Whole wheat is the main ingredient in a range of staple distinct on levels of free phenolic acids; for example, the levels foods that form a part of a healthy diet [4]. Regular intake of of free phenolicacids in 26 wheat genotypes grownin Hungary 2 Journal of Nutrition and Metabolism in three consecutive crop years were largely influenced by the expected that hydrolysis products of a variety of cell wall effect of environment. On the other hand, levels of bound polysaccharides in the fiber fraction may be present in the phenolic acids were stable throughout the growing seasons bound phenolic extracts and that these compounds could and were mostly determined by genetics [15]. bind a fraction of the phenolics to render them inactive in In earlier studies under strictly controlled growth con- the inhibition assays [18]. ditions, we had observed strong positive correlations be- tween growing temperatures, levels of phenolic acids, and 2.5. HPLC-PDAAnalysis ofFree andBoundExtracts. Phenolic the inhibition of glucose uptake into Caco-2 cell monolayers acids of the free and bound fractions were identified by exhibited by extracts from whole wheat (unpublished data). a reverse-phased performance liquid chromatography sys- Here, we investigated if such correlations can be observed tem (Waters 2695, Milford, MA, USA) equipped with for wheat grown under field conditions. a photodiode array detector (PDA) (Waters 996) and autosampler (717 plus, Waters, Milford, MA, USA) as 2. Materials and Methods described by Shamloo et al. [18]. 2.1. Wheat Genotypes. Eight western Canadian wheat ge- notypes (Triticum spp.), representing different commercial 2.6. Glucose Uptake Inhibition Activity Assays classes with different qualities, including AC Corrine, AC Barrie, AC Crystal and Carberry, Snowbird, AC Andrew 2.6.1. Inhibition of Glucose Uptake into Caco-2E Enterocyte (Triticum aestivum L.), AC Navigator and Strongfield durum Monolayers. *e ability of wheat phenolic acid extracts to wheat grains (Triticum turgidum L. var. durum). *ese eight reduce cellular glucose uptake was investigated through the genotypes were grown at three locations over two consec- modified method described by Kwon et al. [10]. *e model of utive crop years (2010 and 2011). confluent Caco-2 cell monolayers grown on 96-well plates was chosen because these previous studies indicated low bio- availability of dietary phenols; therefore, the main inhibitory 2.2.Locations. *ree locations were selected to represent the action was expected to affect apical rather than basolateral wheat growing conditions of the Canadian Prairies. *ese transporters, which is captured by the chosen model. were the Cereal Grain Research Centers of the Lethbridge To initiate the experiment, the confluent Caco-2 cell (Alberta (AB)), Indian Head (Saskatchewan (SK)), and monolayers were rinsed 3 times with PBS and incubated in Portage la Prairie (Manitoba (MB)). preincubation buffer (HEPES buffer with 5mM glucose) for Environmental data conditions were obtained from Sta- 30 minutes at 37 C. Transport experiments were initiated by tistics Canada [16]. Location characteristics and soil compo- replacing the buffer with 100 µL HEPES buffer (pH 7.4 and sition data were obtained from Agriculture and Agri-Food glucose free), containing [ H] 2-deoxyglucose (5mM in Canada [17]. *e mean temperature was lower in 2010 com- glucose-free HEPES buffer) and wheat extracts. *e cells pared to 2011 across all locations, whereas total precipitation were incubated at room temperature for 15 minutes in the was higher in 2010 compared to 2011 across all locations. dark, and the transport experiment was stopped by adding 100 µL of ice-cold preincubation buffer immediately after removal of transport buffer. Cells were washed with 100 µL 2.3. Materials. Phenolic acids standards were purchased of preincubation buffer and then lysed with 60 µL lysis buffer from Sigma-Aldrich (Milwaukee, Wisconsin, USA). All acids (20mg SDS in 1mL 0.2M NaOH) and incubated at room and organic solvents were obtained from Fisher Scientific temperature for 1 hour. 45 µL aliquot of cell lysates was (Whitby, Ontario, Canada). All chemicals used were of added to 5ml scintillation cocktail, and the [ H] 2-deoxy- analytical grade. glucose concentration was quantified by scintillation spec- trometry. Protein content of the remaining cell lysates was 2.4. Preparation of Free and Bound Phenolic Acid Extracts. *e determined using DC Protein Assay Kit (Bio-Rad, USA). *e whole wheat samples were prepared as explained in a pre- glucose uptake into early passages of Caco-2 cells (Caco-2E, vious report [18]. Briefly, they were milled and passed passages 35–47) was expressed as counts per minute beta through a 0.5mm sieve screen using 14,000rpm. *e fine (cpma) per mg protein. Viability of cells and validity of the flour from each sample was stored at−20 C in the dark until assay were demonstrated by the linearity of the uptake rates further processing. Free and bound phenolic acid extractions of glucose in the absence of wheat extracts. were performed using liquid-liquid extraction and alkaline hydrolysis steps [19] as described before [18]. *ese extracts of free or bond phenolic acids were used in the cell culture 2.7.StatisticalAnalysis. Allcellculturedatarepresentmeansof experiments without further purification in order to keep three independent experiments (three sets on different days), sample degradation minimum and to mimic composition in where three parallel transport experiments using three cell the intestinal tract. It is expected that these extracts contain culture wells were performed. All data were analyzed using one- some nonpolar compounds other than the phenolic acids. It way analysis of variance (ANOVA) on a Minitab 14 Statistical is expected that the amount of these compounds in the free Software (Minitab Inc., State college, PA, USA). Sample means phenolic extracts is low and would not interfere with the were compared using Tukey HSD method, and significant analysis of the glucose inhibition. However, it can be differences were considered when P<0.05. Correlations Journal of Nutrition and Metabolism 3 Table 1: Free phenolic acid contents (microgram per gram of dry matter) in the whole grain of 8 wheat varieties grown in three locations in 2010 and 2011 crop years. Growing locations Genotype Year MB SK AB A A A 2010 13.93±0.98 11.56±1.27 11.08±1.09 AC Corrine B B A 2011 25.11±2.92 21.68±2.65 11.16±1.82 A A A 2010 11.19±1.07 11.14±1.67 11.10±2.93 AC Navigator B B B 2011 16.13±1.81 24.07±2.94 16.53±2.87 A A A 2010 23.91±1.73 16.79±2.90 11.17±2.43 Snowbird A A B 2011 23.98±1.09 20.28±2.31 21.79±0.95 A A A 2010 14.75±1.60 18.34±1.01 16.62±0.09 AC Andrew A B B 2011 14.34±0.62 13.81±1.21 11.24±1.01 A A A 2010 18.93±3.2 11.10±0.29 16.31±2.07 Carberry A A B 2011 16.72±1.9 11.15±2.35 16.55±1.98 A A A 2010 4.68±0.63 11.14±2.85 11.49±0.93 AC Crystal B A B 2011 24.03±1.01 11.42±2.39 21.43±1.02 A A A 2010 11.12±1.29 11.06±0.93 24.83±2.87 AC Barrie B B B 2011 18.51±2.76 18.92±2.01 40.98±2.81 A A A 2010 4.56±2.9 11.03±1.76 20.34±2.09 Strongfield B B A 2011 20.91±1.87 21.33±0.98 21.29±2.76 A, B Values are given as mean±SD from duplicate determinations. Different superscript capital letters in the same column in the same dependent variable indicate significant difference (P<0.05). MB�Manitoba; SK�Saskatchewan; AB�Alberta. Table 2: Bound phenolic acid contents (microgram per gram of dry matter) in the whole grain of 8 wheat varieties grown in three locations in 2010 and 2011 crop years. Growing locations Genotype Year MB SK AB A A A 2010 537.41±5.23 555.51±5.44 607.17±9.19 AC Corrine B B B 2011 519.54±6.31 489.23±3.33 582.34±8.32 A A A 2010 366.11±5.03 463.47±4.65 641.6±9.03 AC Navigator B B B 2011 522.78±7.42 422.61±3.98 467.28±6.17 A A A 2010 471.71±6.54 450.01±5.87 528.61±5.54 Snowbird A A B 2011 488.32±7.30 487.24±2.63 483.01±4.59 A A A 2010 492.97±5.20 521.54±4.93 486.34±8.98 AC Andrew A B B 2011 495.96±4.82 461.28±5.75 457.65±6.46 A A A 2010 421.35±7.17 551.45±5.43 600.87±7.34 Carberry B B B 2011 544.86±8.43 472.96±4.54 481.57±8.22 A A A 2010 481.24±9.32 509.15±6.38 690.12±7.56 AC Crystal B B B 2011 415.12±6.82 545.49±10.43 629.27±6.41 A A A 2010 560.98±8.38 417.86±7.77 414.11±5.89 AC Barrie B B B 2011 433.89±5.16 547.56±6.09 528.81±7.94 A A A 2010 459.11±6.32 430.32±5.98 566.96±6.89 Strongfield A B A 2011 458.68±5.22 509.01±8.89 525.81±3.37 A, B Values are given as mean±SD from duplicate determinations. Different superscript capital letters in the same column in the same dependent variable indicate significant difference (P<0.05). MB�Manitoba; SK�Saskatchewan; AB�Alberta. between wheat extract phenolic acid and flavonoid contents and 2010 and 2011 ranged from 4.56 microgram per gram of dry inhibition capacity were done by Pearson’s correlation test. matter (μg/g·dm) in Strongfield grown in Manitoba (MB) in 2010 to 40.98 μg/g·dm in AC Barrie grown in Alberta (AB) in 2011. Contents of free PA were lower in 2010 than in 2011 3. Results and Discussion for all genotypes except for AC Andrew (Table 1). Generally, 3.1. Genotype and Environmental Variation Influence on wheat grown in AB in 2011 produced higher amounts of free Phenolic Acids Levels. Phenolic acid (PA) contents in the PA; specifically, AC Barrie grown in AB contained the free and bound fractions of the eight wheat varieties grown highest amount of free PA (40.98 μg/g·dm) of all genotypes. at different locations are listed in Tables 1 and 2, respectively. Bound PA levels of all genotypes grown during 2010 and Free PA contents across all wheat genotypes grown during 2011 ranged from 366.1 μg/g dm (AC Navigator, MB, 2010) 4 Journal of Nutrition and Metabolism 60 60 50 50 40 40 30 30 AA A 20 20 10 10 0 0 AC corrine AC corrine AC corrine Navigator Navigator Navigator MB SK AB MB SK AB (a) (b) 60 60 50 50 40 40 30 30 20 20 10 10 0 0 Snowbird Snowbird Snowbird AC andrew AC andrew AC andrew MB SK AB MB SK AB (c) (d) 60 60 50 50 B B 40 40 A BB 30 30 20 20 10 10 0 0 Carberry Carberry Carberry AC crystal AC crystal AC crystal MB SK AB MB SK AB (e) (f) 60 60 50 50 40 40 30 30 A B 20 20 10 10 0 0 AC barrie AC barrie AC barrie Strongfield Strongfield Strongfield MB SK AB MB SK AB (g) (h) Figure 1: Relative inhibition of the uptake of [ H] 2-deoxyglucose into CaCo-2 monolayers caused by extracts of free phenolic acids A,B,C obtained from eight wheat genotypes (a–h) grown in MB, SK, and AB over 2010 crop year. Di‡erent capital letter superscripts indicate signiˆcant di‡erences (P < 0.05). Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Journal of Nutrition and Metabolism 5 60 60 50 50 A A 40 40 30 30 20 20 10 10 0 0 AC corrine AC corrine AC corrine Navigator Navigator Navigator MB SK AB MB SK AB (a) (b) 60 60 50 50 40 40 30 30 20 20 10 10 0 0 Snowbird Snowbird Snowbird AC andrew AC andrew AC andrew MB SK AB MB SK AB (c) (d) 60 60 50 50 40 40 30 30 20 20 10 10 0 0 Carberry Carberry Carberry AC crystal AC crystal AC crystal MB SK AB MB SK AB (e) (f) 60 60 50 50 A A 40 40 30 30 20 20 10 10 0 0 AC barrie AC barrie AC barrie Strongfield Strongfield Strongfield MB SK AB MB SK AB (g) (h) Figure 2: Relative inhibition of the uptake of [ H] 2-deoxyglucose into CaCo-2 monolayers caused by extracts of free phenolic acids A,B,C obtained from eight wheat genotypes (a–h) grown in MB, SK, and AB over 2011 crop year. Di‡erent capital letter superscripts indicate signiˆcant di‡erences (P < 0.05). Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) 6 Journal of Nutrition and Metabolism 60 60 50 50 0 0 020 40 60 0 200 400 600 800 . . Sum of free phenolic acids (µg/g dm) Sum of bound phenolic acids (µg/g dm) y = 1.6637x + 4.5193 y = 0.0547x – 19.864 2 2 R = 0.9032 R = 0.891 (a) (b) Figure 3: Correlation between the relative inhibition of glucose uptake into CaCo-2 monolayers and free phenolic acid (a) and bound phenolic acid (b) contents in extracts of eight wheat genotypes grown in MB, SK, and AB over 2011 crop year. to 690.12 μg/g dm (AC Crystal, AB, 2010). AC Crystal, AC Bound phenolic acid extracts of all wheat genotypes Corrine, and AC Navigator (641.6 μg/g·dm) grown in AB in grown in MB, SK, and AB in 2010 and 2011 also inhibited the 2010 and 2011 contained the highest amounts of bound PA, uptake of glucose in confluent Caco-2E monolayers, as shown respectively. in Figures 4(a)–4(h) and Figures 5(a)–5(h), respectively. *e Similar to previously reported data, the contribution of highest inhibitory potencies (21.22% and 16.78%) were ob- the genotype [20] on free phenolic acid levels in whole wheat served for the extracts from AC Crystal and AC Navigator is surpassed by the influence of environmental conditions grown in AB in 2010 (Figures 4(f) and 4(b)), respectively. *e [15, 21] reflected by the yearly variance in the presented data. degree of glucose uptake inhibitions positively correlated with Bound phenolic acid levels were also more influenced by the content of bound phenolic acids, as depicted in Figure 3(b) environment and less by genotype. However, bound PA in (R � 0.891; P<0.05). some genotypes (AC Andrew and Snowbird) showed lower Extracts of free and bound phenolic acids inhibited sensitivity to environmental shifts being more stable in glucose uptake in this Caco-2 model; however, the potency of composition. *is was in line with the results of Mpofu et al. the free phenolic acid extracts exceeded the potency of the [15]. *e values reported for whole wheat bread correspond extracts of bound phenolic acids. Free phenolic acids are fully well with our data, where the highest level of total bound bioaccessible, and some are bioavailable in the small intestine phenolic acids was 690 μg/g. [22], while the bound phenolics are bound to the molecules of the fiber fraction and not fully bioaccessible. *is might be reflected in our model, where we used extracts low in in- 3.2. Effect of Free and Bound Phenolic Acids on Glucose terfering fiber components in the free phenolic fraction where Uptake. Free phenolic acid extracts of all whole wheat va- they exhibited their full potency. In contrast, the presence of rieties grown at MB, SK, and AB over 2010 and 2011 inhibited hydrolyzed fiber constituents in the bound fraction could glucose uptake in confluent Caco-2E monolayers as shown in explain the reduced potency observed for these extracts. Figures 1(a)–1(h) and Figures 2(a)–2(h), respectively. For the However, phenolic acids from both fractions can contribute 2010 crop year, extracts from AC Barrie grown in AB showed to a blunting of cellular glucose uptake into small intestinal the highest inhibition (46.18%) (Figure 1(g)), followed by cells and therefore could contribute to the dampening of Snowbird grown in MB (45.12%) (Figure 1(c)). For the 2011 postprandial hyperglycemia. crop year, again extracts from AC Barrie grown in AB showed *e concentrations of total flavonoids in the glucose the highest inhibitory potency (56.32%) (Figure 2(g)), fol- inhibition assays ranged between 0.5 and 2 µg/ml for free lowed by AC Corrine grown in MB (48.62%) (Figure 2(a)). phenolics and 35–50 µg/ml for bound phenolics. Assuming *e free phenolic acid content of all wheat genotypes posi- an average molecular mass of 200g/mol, the highest con- tively correlated with the degree of inhibition of glucose centration of total free phenolic acids would have been uptake, as shown in Figure 3(a) (R � 0.903; P<0.05). 10 µmol/l and from bound phenolic acid 250 µmol/l. Glucose uptake inhibition (%) Glucose uptake inhibition (%) Journal of Nutrition and Metabolism 7 25 25 20 20 15 15 10 10 5 5 0 0 AC corrine AC corrine AC corrine Navigator Navigator Navigator MB SK AB MB SK AB (a) (b) 25 25 20 20 15 15 10 10 B A 5 5 0 0 Snowbird Snowbird Snowbird AC andrew AC andrew AC andrew MB SK AB MB SK AB (c) (d) 25 25 20 20 15 15 10 10 B 5 5 0 0 Carberry Carberry Carberry AC crystal AC crystal AC crystal MB SK AB MB SK AB (e) (f) 25 25 20 20 15 15 10 10 5 5 0 0 AC barrie AC barrie AC barrie Strongfield Strongfield Strongfield MB SK AB MB SK AB (g) (h) Figure 4: Relative inhibition of the uptake of [ H] 2-deoxyglucose into CaCo-2 monolayers caused by extracts of bond phenolic acids A,B,C obtained from eight wheat genotypes (a–h) grown in MB, SK, and AB over 2010 crop year. Di‡erent capital letter superscripts indicate signiˆcant di‡erences (P < 0.05). Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) 8 Journal of Nutrition and Metabolism 25 25 20 20 15 15 10 10 5 5 0 0 AC corrine AC corrine AC corrine Navigator Navigator Navigator MB SK AB MB SK AB (a) (b) 25 25 20 20 15 15 10 10 A B 5 5 0 0 Snowbird Snowbird Snowbird AC andrew AC andrew AC andrew MB SK AB MB SK AB (c) (d) 25 25 20 20 15 15 C B 10 10 5 5 0 0 Carberry Carberry Carberry AC crystal AC crystal AC crystal MB SK AB MB SK AB (e) (f) 25 25 20 20 15 15 10 10 5 5 0 0 AC barrie AC barrie AC barrie Strongfield Strongfield Strongfield MB SK AB MB SK AB (g) (h) Figure 5: Relative inhibition of the uptake of [ H] 2-deoxyglucose into CaCo-2 monolayers caused by extracts of bond phenolic acids A,B,C obtained from eight wheat genotypes (a–h) grown in MB, SK, and AB over 2011 crop year. Di‡erent capital letter superscripts indicate signiˆcant di‡erences (P < 0.05). Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Journal of Nutrition and Metabolism 9 Assuming a stomach volume of one liter, the intake of 2mg References free phenolic acid or 35mg of bound phenolic acids would [1] World Health Organization, Global Report on Diabetes, 2016, achieve the concentrations used in our glucose inhibition http://apps.who.int/iris/bitstream/10665/204871/1/9789241565257_ assays. *e most abundant compound in both free and eng.pdf. bound phenolic acid extracts in wheat is ferulic acid, and it [2] American Diabetes Association, “Diagnosis and classification has been suggested that a daily dietary uptake of about of diabetes mellitus,” Diabetes Care, vol. 32, no. 1, pp. S62–S67, 77mg ferulic acid may suppress hyperglycemia [22]. 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Inhibition of Intestinal Cellular Glucose Uptake by Phenolics Extracted from Whole Wheat Grown at Different Locations

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Copyright © 2018 Maryam Shamloo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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10.1155/2018/5421714
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Abstract

Hindawi Journal of Nutrition and Metabolism Volume 2018, Article ID 5421714, 10 pages https://doi.org/10.1155/2018/5421714 Research Article Inhibition of Intestinal Cellular Glucose Uptake by Phenolics Extracted from Whole Wheat Grown at Different Locations 1,2 1,2 3 Maryam Shamloo , Peter J. H. Jones, and Peter K. Eck Richardson Centre for Functional Foods and Nutraceuticals, University of Manitoba, Winnipeg, MB, Canada R3T 2N2 Department of Food Science, University of Manitoba, Winnipeg, MB, Canada R3T 2N2 Department of Human Nutritional Sciences, University of Manitoba, W569 Duff Roblin Building, 190 Dysart Road, Winnipeg, MB, Canada R3T 2N2 Correspondence should be addressed to Peter K. Eck; peter.eck@umanitoba.ca Received 7 November 2017; Accepted 28 December 2017; Published 18 March 2018 Academic Editor: Stan Kubow Copyright © 2018 Maryam Shamloo et al. *is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Whole grain consumption is associated with reduced risk of type 2 diabetes, and the underlying mechanism might be related to the actions of polyphenols. Dietary polyphenols contribute to low glycemic indices through inhibition of intestinal glucose transport proteins. *is study has two objectives: (1) to evaluate how the contents of phenolic acids in wheat vary by genetic background and growth condition and (2) to evaluate how these changes translate into physiologic relevance by investigating cellular glucose transporter inhibitions. Phenolic acids were extracted from wheat varieties grown at different locations over two crop years. *e degree of inhibition of glucose uptake into human Caco-2E cells was determined. Free and bound phenolic acid extracts of all wheat genotypes inhibited glucose uptake. Degree of glucose uptake inhibitions positively correlated with the contents of free and 2 2 bound phenolic acids, and the correlation coefficients were R � 0.91 and R � 0.89, respectively. Genotype and environment influenced the content of free and bound phenolic acids which linearly translated to the degree of glucose uptake inhibition in a model of intestinal absorption (P <0.05). Results of this work mechanistically support the hypothesis that dietary phenols positively influence the glycemic index and therefore the health properties of whole grain consumption. whole grain products is associated with a 20–30% reduction 1. Introduction in the risk of type 2 diabetes [5, 6]. For example, whole wheat Diabetes mellitus has become an emerging global health breads generate lower glycemic indices than white bread [7], problem. Currently, estimated 422 million adults are suf- which is in part attributed to polyphenols found in the bran fering from diabetes and that number will increase to 642 fraction of whole wheat [8]. *ese polyphenols are known to million by 2040 [1]. Type 2 diabetes is characterized by the be efficient in lowering blood glucose levels [9]; however, the body’s insensitivity to insulin and is characterized by increased mechanisms remain largely undetermined [6]. Some plant blood sugar levels [2]. In diabetic patients, the expression of bioactives inhibit intestinal glucose transporters [10, 11] intestinal glucose transporters has been reported to be 3- to causing a blunted glucose absorption [6, 7, 12]. 4-fold higher than healthy controls [3], and therefore, higher Wheat genetics and growing environments play a critical amount of glucose will be absorbed by these patients in role in influencing the accumulation of secondary plant a shorter period of time, leading to increased postprandial metabolites [13], including polyphenols. *e importance of the glycemia. *us, an effective treatment option for diabetes and genotype is demonstrated by the fact that winter wheat va- diabetes-related complications is to dampen or inhibit in- rieties contain twice the levels of total phenolic acids than testinal glucose transporters and/or glucose absorption. other genotypes [14]. *e impact of the environment is very Whole wheat is the main ingredient in a range of staple distinct on levels of free phenolic acids; for example, the levels foods that form a part of a healthy diet [4]. Regular intake of of free phenolicacids in 26 wheat genotypes grownin Hungary 2 Journal of Nutrition and Metabolism in three consecutive crop years were largely influenced by the expected that hydrolysis products of a variety of cell wall effect of environment. On the other hand, levels of bound polysaccharides in the fiber fraction may be present in the phenolic acids were stable throughout the growing seasons bound phenolic extracts and that these compounds could and were mostly determined by genetics [15]. bind a fraction of the phenolics to render them inactive in In earlier studies under strictly controlled growth con- the inhibition assays [18]. ditions, we had observed strong positive correlations be- tween growing temperatures, levels of phenolic acids, and 2.5. HPLC-PDAAnalysis ofFree andBoundExtracts. Phenolic the inhibition of glucose uptake into Caco-2 cell monolayers acids of the free and bound fractions were identified by exhibited by extracts from whole wheat (unpublished data). a reverse-phased performance liquid chromatography sys- Here, we investigated if such correlations can be observed tem (Waters 2695, Milford, MA, USA) equipped with for wheat grown under field conditions. a photodiode array detector (PDA) (Waters 996) and autosampler (717 plus, Waters, Milford, MA, USA) as 2. Materials and Methods described by Shamloo et al. [18]. 2.1. Wheat Genotypes. Eight western Canadian wheat ge- notypes (Triticum spp.), representing different commercial 2.6. Glucose Uptake Inhibition Activity Assays classes with different qualities, including AC Corrine, AC Barrie, AC Crystal and Carberry, Snowbird, AC Andrew 2.6.1. Inhibition of Glucose Uptake into Caco-2E Enterocyte (Triticum aestivum L.), AC Navigator and Strongfield durum Monolayers. *e ability of wheat phenolic acid extracts to wheat grains (Triticum turgidum L. var. durum). *ese eight reduce cellular glucose uptake was investigated through the genotypes were grown at three locations over two consec- modified method described by Kwon et al. [10]. *e model of utive crop years (2010 and 2011). confluent Caco-2 cell monolayers grown on 96-well plates was chosen because these previous studies indicated low bio- availability of dietary phenols; therefore, the main inhibitory 2.2.Locations. *ree locations were selected to represent the action was expected to affect apical rather than basolateral wheat growing conditions of the Canadian Prairies. *ese transporters, which is captured by the chosen model. were the Cereal Grain Research Centers of the Lethbridge To initiate the experiment, the confluent Caco-2 cell (Alberta (AB)), Indian Head (Saskatchewan (SK)), and monolayers were rinsed 3 times with PBS and incubated in Portage la Prairie (Manitoba (MB)). preincubation buffer (HEPES buffer with 5mM glucose) for Environmental data conditions were obtained from Sta- 30 minutes at 37 C. Transport experiments were initiated by tistics Canada [16]. Location characteristics and soil compo- replacing the buffer with 100 µL HEPES buffer (pH 7.4 and sition data were obtained from Agriculture and Agri-Food glucose free), containing [ H] 2-deoxyglucose (5mM in Canada [17]. *e mean temperature was lower in 2010 com- glucose-free HEPES buffer) and wheat extracts. *e cells pared to 2011 across all locations, whereas total precipitation were incubated at room temperature for 15 minutes in the was higher in 2010 compared to 2011 across all locations. dark, and the transport experiment was stopped by adding 100 µL of ice-cold preincubation buffer immediately after removal of transport buffer. Cells were washed with 100 µL 2.3. Materials. Phenolic acids standards were purchased of preincubation buffer and then lysed with 60 µL lysis buffer from Sigma-Aldrich (Milwaukee, Wisconsin, USA). All acids (20mg SDS in 1mL 0.2M NaOH) and incubated at room and organic solvents were obtained from Fisher Scientific temperature for 1 hour. 45 µL aliquot of cell lysates was (Whitby, Ontario, Canada). All chemicals used were of added to 5ml scintillation cocktail, and the [ H] 2-deoxy- analytical grade. glucose concentration was quantified by scintillation spec- trometry. Protein content of the remaining cell lysates was 2.4. Preparation of Free and Bound Phenolic Acid Extracts. *e determined using DC Protein Assay Kit (Bio-Rad, USA). *e whole wheat samples were prepared as explained in a pre- glucose uptake into early passages of Caco-2 cells (Caco-2E, vious report [18]. Briefly, they were milled and passed passages 35–47) was expressed as counts per minute beta through a 0.5mm sieve screen using 14,000rpm. *e fine (cpma) per mg protein. Viability of cells and validity of the flour from each sample was stored at−20 C in the dark until assay were demonstrated by the linearity of the uptake rates further processing. Free and bound phenolic acid extractions of glucose in the absence of wheat extracts. were performed using liquid-liquid extraction and alkaline hydrolysis steps [19] as described before [18]. *ese extracts of free or bond phenolic acids were used in the cell culture 2.7.StatisticalAnalysis. Allcellculturedatarepresentmeansof experiments without further purification in order to keep three independent experiments (three sets on different days), sample degradation minimum and to mimic composition in where three parallel transport experiments using three cell the intestinal tract. It is expected that these extracts contain culture wells were performed. All data were analyzed using one- some nonpolar compounds other than the phenolic acids. It way analysis of variance (ANOVA) on a Minitab 14 Statistical is expected that the amount of these compounds in the free Software (Minitab Inc., State college, PA, USA). Sample means phenolic extracts is low and would not interfere with the were compared using Tukey HSD method, and significant analysis of the glucose inhibition. However, it can be differences were considered when P<0.05. Correlations Journal of Nutrition and Metabolism 3 Table 1: Free phenolic acid contents (microgram per gram of dry matter) in the whole grain of 8 wheat varieties grown in three locations in 2010 and 2011 crop years. Growing locations Genotype Year MB SK AB A A A 2010 13.93±0.98 11.56±1.27 11.08±1.09 AC Corrine B B A 2011 25.11±2.92 21.68±2.65 11.16±1.82 A A A 2010 11.19±1.07 11.14±1.67 11.10±2.93 AC Navigator B B B 2011 16.13±1.81 24.07±2.94 16.53±2.87 A A A 2010 23.91±1.73 16.79±2.90 11.17±2.43 Snowbird A A B 2011 23.98±1.09 20.28±2.31 21.79±0.95 A A A 2010 14.75±1.60 18.34±1.01 16.62±0.09 AC Andrew A B B 2011 14.34±0.62 13.81±1.21 11.24±1.01 A A A 2010 18.93±3.2 11.10±0.29 16.31±2.07 Carberry A A B 2011 16.72±1.9 11.15±2.35 16.55±1.98 A A A 2010 4.68±0.63 11.14±2.85 11.49±0.93 AC Crystal B A B 2011 24.03±1.01 11.42±2.39 21.43±1.02 A A A 2010 11.12±1.29 11.06±0.93 24.83±2.87 AC Barrie B B B 2011 18.51±2.76 18.92±2.01 40.98±2.81 A A A 2010 4.56±2.9 11.03±1.76 20.34±2.09 Strongfield B B A 2011 20.91±1.87 21.33±0.98 21.29±2.76 A, B Values are given as mean±SD from duplicate determinations. Different superscript capital letters in the same column in the same dependent variable indicate significant difference (P<0.05). MB�Manitoba; SK�Saskatchewan; AB�Alberta. Table 2: Bound phenolic acid contents (microgram per gram of dry matter) in the whole grain of 8 wheat varieties grown in three locations in 2010 and 2011 crop years. Growing locations Genotype Year MB SK AB A A A 2010 537.41±5.23 555.51±5.44 607.17±9.19 AC Corrine B B B 2011 519.54±6.31 489.23±3.33 582.34±8.32 A A A 2010 366.11±5.03 463.47±4.65 641.6±9.03 AC Navigator B B B 2011 522.78±7.42 422.61±3.98 467.28±6.17 A A A 2010 471.71±6.54 450.01±5.87 528.61±5.54 Snowbird A A B 2011 488.32±7.30 487.24±2.63 483.01±4.59 A A A 2010 492.97±5.20 521.54±4.93 486.34±8.98 AC Andrew A B B 2011 495.96±4.82 461.28±5.75 457.65±6.46 A A A 2010 421.35±7.17 551.45±5.43 600.87±7.34 Carberry B B B 2011 544.86±8.43 472.96±4.54 481.57±8.22 A A A 2010 481.24±9.32 509.15±6.38 690.12±7.56 AC Crystal B B B 2011 415.12±6.82 545.49±10.43 629.27±6.41 A A A 2010 560.98±8.38 417.86±7.77 414.11±5.89 AC Barrie B B B 2011 433.89±5.16 547.56±6.09 528.81±7.94 A A A 2010 459.11±6.32 430.32±5.98 566.96±6.89 Strongfield A B A 2011 458.68±5.22 509.01±8.89 525.81±3.37 A, B Values are given as mean±SD from duplicate determinations. Different superscript capital letters in the same column in the same dependent variable indicate significant difference (P<0.05). MB�Manitoba; SK�Saskatchewan; AB�Alberta. between wheat extract phenolic acid and flavonoid contents and 2010 and 2011 ranged from 4.56 microgram per gram of dry inhibition capacity were done by Pearson’s correlation test. matter (μg/g·dm) in Strongfield grown in Manitoba (MB) in 2010 to 40.98 μg/g·dm in AC Barrie grown in Alberta (AB) in 2011. Contents of free PA were lower in 2010 than in 2011 3. Results and Discussion for all genotypes except for AC Andrew (Table 1). Generally, 3.1. Genotype and Environmental Variation Influence on wheat grown in AB in 2011 produced higher amounts of free Phenolic Acids Levels. Phenolic acid (PA) contents in the PA; specifically, AC Barrie grown in AB contained the free and bound fractions of the eight wheat varieties grown highest amount of free PA (40.98 μg/g·dm) of all genotypes. at different locations are listed in Tables 1 and 2, respectively. Bound PA levels of all genotypes grown during 2010 and Free PA contents across all wheat genotypes grown during 2011 ranged from 366.1 μg/g dm (AC Navigator, MB, 2010) 4 Journal of Nutrition and Metabolism 60 60 50 50 40 40 30 30 AA A 20 20 10 10 0 0 AC corrine AC corrine AC corrine Navigator Navigator Navigator MB SK AB MB SK AB (a) (b) 60 60 50 50 40 40 30 30 20 20 10 10 0 0 Snowbird Snowbird Snowbird AC andrew AC andrew AC andrew MB SK AB MB SK AB (c) (d) 60 60 50 50 B B 40 40 A BB 30 30 20 20 10 10 0 0 Carberry Carberry Carberry AC crystal AC crystal AC crystal MB SK AB MB SK AB (e) (f) 60 60 50 50 40 40 30 30 A B 20 20 10 10 0 0 AC barrie AC barrie AC barrie Strongfield Strongfield Strongfield MB SK AB MB SK AB (g) (h) Figure 1: Relative inhibition of the uptake of [ H] 2-deoxyglucose into CaCo-2 monolayers caused by extracts of free phenolic acids A,B,C obtained from eight wheat genotypes (a–h) grown in MB, SK, and AB over 2010 crop year. Di‡erent capital letter superscripts indicate signiˆcant di‡erences (P < 0.05). Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Journal of Nutrition and Metabolism 5 60 60 50 50 A A 40 40 30 30 20 20 10 10 0 0 AC corrine AC corrine AC corrine Navigator Navigator Navigator MB SK AB MB SK AB (a) (b) 60 60 50 50 40 40 30 30 20 20 10 10 0 0 Snowbird Snowbird Snowbird AC andrew AC andrew AC andrew MB SK AB MB SK AB (c) (d) 60 60 50 50 40 40 30 30 20 20 10 10 0 0 Carberry Carberry Carberry AC crystal AC crystal AC crystal MB SK AB MB SK AB (e) (f) 60 60 50 50 A A 40 40 30 30 20 20 10 10 0 0 AC barrie AC barrie AC barrie Strongfield Strongfield Strongfield MB SK AB MB SK AB (g) (h) Figure 2: Relative inhibition of the uptake of [ H] 2-deoxyglucose into CaCo-2 monolayers caused by extracts of free phenolic acids A,B,C obtained from eight wheat genotypes (a–h) grown in MB, SK, and AB over 2011 crop year. Di‡erent capital letter superscripts indicate signiˆcant di‡erences (P < 0.05). Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) 6 Journal of Nutrition and Metabolism 60 60 50 50 0 0 020 40 60 0 200 400 600 800 . . Sum of free phenolic acids (µg/g dm) Sum of bound phenolic acids (µg/g dm) y = 1.6637x + 4.5193 y = 0.0547x – 19.864 2 2 R = 0.9032 R = 0.891 (a) (b) Figure 3: Correlation between the relative inhibition of glucose uptake into CaCo-2 monolayers and free phenolic acid (a) and bound phenolic acid (b) contents in extracts of eight wheat genotypes grown in MB, SK, and AB over 2011 crop year. to 690.12 μg/g dm (AC Crystal, AB, 2010). AC Crystal, AC Bound phenolic acid extracts of all wheat genotypes Corrine, and AC Navigator (641.6 μg/g·dm) grown in AB in grown in MB, SK, and AB in 2010 and 2011 also inhibited the 2010 and 2011 contained the highest amounts of bound PA, uptake of glucose in confluent Caco-2E monolayers, as shown respectively. in Figures 4(a)–4(h) and Figures 5(a)–5(h), respectively. *e Similar to previously reported data, the contribution of highest inhibitory potencies (21.22% and 16.78%) were ob- the genotype [20] on free phenolic acid levels in whole wheat served for the extracts from AC Crystal and AC Navigator is surpassed by the influence of environmental conditions grown in AB in 2010 (Figures 4(f) and 4(b)), respectively. *e [15, 21] reflected by the yearly variance in the presented data. degree of glucose uptake inhibitions positively correlated with Bound phenolic acid levels were also more influenced by the content of bound phenolic acids, as depicted in Figure 3(b) environment and less by genotype. However, bound PA in (R � 0.891; P<0.05). some genotypes (AC Andrew and Snowbird) showed lower Extracts of free and bound phenolic acids inhibited sensitivity to environmental shifts being more stable in glucose uptake in this Caco-2 model; however, the potency of composition. *is was in line with the results of Mpofu et al. the free phenolic acid extracts exceeded the potency of the [15]. *e values reported for whole wheat bread correspond extracts of bound phenolic acids. Free phenolic acids are fully well with our data, where the highest level of total bound bioaccessible, and some are bioavailable in the small intestine phenolic acids was 690 μg/g. [22], while the bound phenolics are bound to the molecules of the fiber fraction and not fully bioaccessible. *is might be reflected in our model, where we used extracts low in in- 3.2. Effect of Free and Bound Phenolic Acids on Glucose terfering fiber components in the free phenolic fraction where Uptake. Free phenolic acid extracts of all whole wheat va- they exhibited their full potency. In contrast, the presence of rieties grown at MB, SK, and AB over 2010 and 2011 inhibited hydrolyzed fiber constituents in the bound fraction could glucose uptake in confluent Caco-2E monolayers as shown in explain the reduced potency observed for these extracts. Figures 1(a)–1(h) and Figures 2(a)–2(h), respectively. For the However, phenolic acids from both fractions can contribute 2010 crop year, extracts from AC Barrie grown in AB showed to a blunting of cellular glucose uptake into small intestinal the highest inhibition (46.18%) (Figure 1(g)), followed by cells and therefore could contribute to the dampening of Snowbird grown in MB (45.12%) (Figure 1(c)). For the 2011 postprandial hyperglycemia. crop year, again extracts from AC Barrie grown in AB showed *e concentrations of total flavonoids in the glucose the highest inhibitory potency (56.32%) (Figure 2(g)), fol- inhibition assays ranged between 0.5 and 2 µg/ml for free lowed by AC Corrine grown in MB (48.62%) (Figure 2(a)). phenolics and 35–50 µg/ml for bound phenolics. Assuming *e free phenolic acid content of all wheat genotypes posi- an average molecular mass of 200g/mol, the highest con- tively correlated with the degree of inhibition of glucose centration of total free phenolic acids would have been uptake, as shown in Figure 3(a) (R � 0.903; P<0.05). 10 µmol/l and from bound phenolic acid 250 µmol/l. Glucose uptake inhibition (%) Glucose uptake inhibition (%) Journal of Nutrition and Metabolism 7 25 25 20 20 15 15 10 10 5 5 0 0 AC corrine AC corrine AC corrine Navigator Navigator Navigator MB SK AB MB SK AB (a) (b) 25 25 20 20 15 15 10 10 B A 5 5 0 0 Snowbird Snowbird Snowbird AC andrew AC andrew AC andrew MB SK AB MB SK AB (c) (d) 25 25 20 20 15 15 10 10 B 5 5 0 0 Carberry Carberry Carberry AC crystal AC crystal AC crystal MB SK AB MB SK AB (e) (f) 25 25 20 20 15 15 10 10 5 5 0 0 AC barrie AC barrie AC barrie Strongfield Strongfield Strongfield MB SK AB MB SK AB (g) (h) Figure 4: Relative inhibition of the uptake of [ H] 2-deoxyglucose into CaCo-2 monolayers caused by extracts of bond phenolic acids A,B,C obtained from eight wheat genotypes (a–h) grown in MB, SK, and AB over 2010 crop year. Di‡erent capital letter superscripts indicate signiˆcant di‡erences (P < 0.05). Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) 8 Journal of Nutrition and Metabolism 25 25 20 20 15 15 10 10 5 5 0 0 AC corrine AC corrine AC corrine Navigator Navigator Navigator MB SK AB MB SK AB (a) (b) 25 25 20 20 15 15 10 10 A B 5 5 0 0 Snowbird Snowbird Snowbird AC andrew AC andrew AC andrew MB SK AB MB SK AB (c) (d) 25 25 20 20 15 15 C B 10 10 5 5 0 0 Carberry Carberry Carberry AC crystal AC crystal AC crystal MB SK AB MB SK AB (e) (f) 25 25 20 20 15 15 10 10 5 5 0 0 AC barrie AC barrie AC barrie Strongfield Strongfield Strongfield MB SK AB MB SK AB (g) (h) Figure 5: Relative inhibition of the uptake of [ H] 2-deoxyglucose into CaCo-2 monolayers caused by extracts of bond phenolic acids A,B,C obtained from eight wheat genotypes (a–h) grown in MB, SK, and AB over 2011 crop year. Di‡erent capital letter superscripts indicate signiˆcant di‡erences (P < 0.05). Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Glucose uptake inhibition (%) Journal of Nutrition and Metabolism 9 Assuming a stomach volume of one liter, the intake of 2mg References free phenolic acid or 35mg of bound phenolic acids would [1] World Health Organization, Global Report on Diabetes, 2016, achieve the concentrations used in our glucose inhibition http://apps.who.int/iris/bitstream/10665/204871/1/9789241565257_ assays. *e most abundant compound in both free and eng.pdf. bound phenolic acid extracts in wheat is ferulic acid, and it [2] American Diabetes Association, “Diagnosis and classification has been suggested that a daily dietary uptake of about of diabetes mellitus,” Diabetes Care, vol. 32, no. 1, pp. S62–S67, 77mg ferulic acid may suppress hyperglycemia [22]. 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Chen et al., “Flavonoid inhibition of sodium-dependent vitamin C transporter 1 (SVCT1) and *e authors declare that they have no conflicts of interest. glucose transporter isoform 2 (GLUT2), intestinal transporters for vitamin C and Glucose,” Journal of Biological Chemistry, Authors’ Contributions vol. 277, no. 18, pp. 15252–15260, 2002. [12] A. Scalbert, C. Morand, C. Manach, and C. Remesy, “Ab- Maryam Shamloo was the principal manuscript author and sorption and metabolism of polyphenols in the gut and impact was responsible for data collection and conducting all the on health,” Biomedicine and pharmacotherapy, vol. 56, no. 6, laboratory analysis including cell culture experiments and pp. 276–282, 2002. statistical analysis. Peter J. H. Jones was responsible for [13] A. Ramakrishna and G. A. 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Beta, “Genotype and environmental variation in phenolic content, phenolic acid Acknowledgments composition, and antioxidant activity of hard spring wheat,” Journal of Agricultural and Food Chemistry, vol. 54, no. 4, *is research was funded in part by Manitoba Science and pp. 1265–1270, 2006. Technology International Collaboration Fund (STIC) and [16] Environment Canada, Historical Data, 2017, http://climate. Food Advancement through Science and Training (FAST) weather.gc.ca/historical_data/search_historic_data_e.html. program. *e authors would like to thank Drs. Susan [17] Agriculture and Agri-Food Canada, >e National Soil Da- Arntfield and Nancy Ames for their assistance with finding taBase (NSDB), 2017, http://sis.agr.gc.ca/cansis/nsdb/index. the wheat samples through Cereal Research Centre, Canadian html. Wheat Board and Agriculture and Agri-Food Canada, and [18] M. Shamloo, E. A. Babawale, A. Furtado, R. J. Henry, Dennis Labossiere, Tracy Exley, Kim Kuzminski, and Camille P. K. 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