Complex Formation of Blueberry (Vaccinium angustifolium

Article pubs.acs.org/JAFC

Complex Formation of Blueberry (Vaccinium angustifolium) Anthocyanins during Freeze-Drying and Its Influence on Their Biological Activity Julieta Correa-Betanzo,†,# Priya Padmanabhan,‡ Milena Corredig,† Jayasankar Subramanian,‡ and Gopinadhan Paliyath*,‡ †

Department of Food Science, and ‡Department of Plant Agriculture, University of Guelph, Guelph, Ontario Canada N1G 2W1 # Plant-Food Molecular Breeding, NutriOmics Group, Instituto Technológico de Estudios Superiores, Monterrey, N.L., Mexico ABSTRACT: Biological activity of polyphenols is influenced by their uptake and is highly influenced by their interactions with the food matrix. This study evaluated the complex formation of blueberry polyphenols with fruit matrixes such as pectin and cellulose and their effect on the biological and antiproliferative properties of human colon cell lines HT-29 and CRL 1790. Free or complexed polyphenols were isolated by dialyzing aqueous or methanolic blueberry homogenates. Seven phenolic compounds and thirteen anthocyanins were identified in blueberry extracts. Blueberry extracts showed varying degrees of antioxidant and antiproliferative activities, as well as α-glucosidase activity. Fruit matrix containing cellulose and pectin, or purified polygalacturonic acid and cellulose, did not retain polyphenols and showed very low antioxidant or antiproliferative activities. These findings suggest that interactions between polyphenols and the food matrix may be more complex than a simple association and may play an important role in the bioefficacy of blueberry polyphenols. KEYWORDS: anthocyanins, flavonoids, food matrix interactions, α-glucosidase, antioxidant activity, antiproliferative activity

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INTRODUCTION Blueberries (Vaccinum angustifolium) are widely grown in eastern Canada and northeastern U.S and are becoming a popular berry fruit because of their high polyphenol content that provides health beneficial properties. In Canada and the United States, most of the wild blueberries are managed and harvested commercially with more than 97% of the total production being processed.1 Dietary polyphenols possess several biological properties including antioxidant activity, ability to modulate signal transduction processes, and the regulation of gene expression, which enable them to provide protection from the development of chronic degenerative diseases such as cardiovascular diseases, type II diabetes, and cancer.2−5 Blueberries possess a wide array of phenolic components and anthocyanins.6,7 Anthocyanins can be present as monoglycosides (e.g., galactoside, glucoside, and arabinosides) of five aglycones (delphinidin, cyanidin, petunidin, peonidin, and malvidin). Since each aglycone may be glycosylated and/or acylated by different sugars, phenolic acids, and aliphatic acids, nearly 539 different anthocyanins have been reported from plants.8 Functionality of foods is usually evaluated based on the levels of individual components and their biological activity; however, potential effects of food matrix on their absorption and biological action are rarely considered.9 In general, it has been observed that anthocyanins are absorbed very poorly in the gastrointestinal system. The influence of food matrix in the absorption of polyphenols, their mode of action, and the influence of microbiome on their metabolism are areas that are currently under investigation. In a previous study, blueberry homogenates subjected to sequential treatments simulating gastric conditions did not alter the polyphenol profiles of © 2015 American Chemical Society

blueberry homogenates, but treatments under intestinal conditions (alkaline pH) and colonic fermentation resulted in a drastic reduction in the polyphenol levels.10 Moreover, several simple phenolic acids were generated by microbial catabolism of the polyphenolic components. However, some ingredients such as the acylated anthocyanins were much more resistant to digestion. Thus, it appeared likely that some of the phenolic components complexed with fruit matrixes may be protected during gastrointestinal transit. Since functionality of foods is highly influenced by the macromolecular organization of food and the interactions between food components, experiments performed on pure components may not fully reflect a realistic situation.11,12 It is essential to understand the influence of the food matrix on the release of the functional components in order to develop value-added products with enhanced health promoting functions. Studies addressing total polyphenols and anthocyanins from complex blueberry mixtures are rare; therefore, the objective of this work was to identify free as well as complexed polyphenols present in blueberries and analyze their in vitro antioxidant capacity and antiproliferative activity using colon-derived normal and cancer cells. Comparing such properties of different polyphenol fractions from blueberries may help to understand the role of polyphenol complexes in nutrition and health. A colon cancer cell line (HT-29) widely employed as a model of colorectal cancer was used along with a normal colon cell line (CRL-1790) to assess and compare the antiproliferative effect of free and complexed Received: Revised: Accepted: Published: 2935

October 2, 2014 February 28, 2015 March 2, 2015 March 2, 2015 DOI: 10.1021/acs.jafc.5b00016 J. Agric. Food Chem. 2015, 63, 2935−2946

Article

Journal of Agricultural and Food Chemistry

polyphenols with methanol, filtered with 0.45 μm nylon filters, and subjected to LC-MS analyses. Identification of Individual Polyphenols by LC- ESI- MS. Chromatographic analysis of anthocyanins isolated from the crude extracts, the dialyzed extracts, and the dialyzate fractions was performed on an Agilent 1100 series LC-MS. Separation was carried out using an X-Terra MS C-18 (5 μm, 150 × 3.0 mm, Waters Corporation, MA). Anthocyanins were eluted with a gradient of mobile phases formed by mixing methanol (phase A) and 2.0% (v/v) formic acid (phase B) at a flow rate of 0.8 mL/min. The gradient used was as follows: 0−2 min, 93% B; 2−30 min, 80% B; 30−45 min, 70% B; 45−50 min; 65% B, 50−60 min, 50% B; 60−65 min, 20% B; 65−70 min, 93% B. Detection was carried out both at 520 and 260 nm. Electrospray ionization (ESI) was performed with an API-ES mass spectrometer. Nitrogen was used as the nebulizing and drying gas, 12 L/min at 350 °C, ion spray voltage, 4000 V, and fragmentor voltage, 80 V. Electrospray ionization mass spectra were scanned from m/z 100 to 1000. Spectra were acquired in the positive ion mode for anthocyanins and negative ion mode for phenolics and flavonoids. A sample injection volume of 20 μL (20 μg) was used for all samples. Chemical identification of the compounds was achieved by matching the molecular ions (m/z) and daughter ions obtained by LC-ESI-MS with spectral data (www.metabolomics.jp). Relative amounts of individual phenolic components were calculated as follows. The sum of all peak areas was taken as 100% resulting from the separation of 20 μg of phenolics sample. Individual peak areas corresponding to specific components were also calculated. From these values, (individual peak area/total peak area) × 20, the relative content of specific peaks in micrograms was obtained. From these values, mg phenolics content per gram fresh weight of fruit could be calculated knowing the fresh weight of the sample and its polyphenol content. Antioxidant Assays. Total antioxidant activity of blueberry crude extracts and dialyzed fractions was measured by using 2,2-diphenyl-1picrylhydrazyl (DPPH) radical (Molyneux),16 superoxide scavenging,17 and hydroxyl radical scavenging18 assays according to standard methods. α-Glucosidase Inhibition. α-Glucosidase inhibitory activity was measured as described.19 Blueberry polyphenol samples were diluted with water to a sample volume of 50 μL to obtain concentrations of 10, 25, 50, and 100 μg/mL of polyphenols, and water of equal volume was used as a control. Reaction was initiated by the addition of 100 μL of 0.1 M sodium phosphate buffer (pH 6.9) containing α-glucosidase solution (1.0 U/mL) to the samples followed by incubation at 25 °C for 10 min. After the reaction, 50 μL of 5 mM p-nitrophenyl-α-Dglucopyranoside solution [prepared in 0.1 M sodium phosphate buffer (pH 6.9)] was added to the reaction mixture (final volume = 200 μL) and incubated at 25 °C for 5 min. After incubation, distilled water (800 μL) was added to the reaction mixture and the absorbance was recorded at λmax = 405 nm (A). Inhibition of the enzyme activity was expressed as percentage inhibition and calculated as α-glucosidase inhibition (%) = (A control − A sample)/(A control) × 100. Fruit Matrix Isolation and Estimation of Pectin Content. Polyphenols were removed from whole blueberries (50 g) by soaking (5×) in 50% v/v ethanol (100 mL) and (2×) in distilled water (100 mL) for 1 h each. The fruits were homogenized in water as described in Polyphenol Extraction. To verify the absence of polyphenols, samples were dialyzed overnight, and Folin−Ciocalteau assay was performed on the extracts. Samples containing the fruit matrix were stored at −20 °C until further analysis. Pectin content in the fruit matrix and crude extracts was estimated according to Taylor.15 Briefly, 200 μL of samples, 3 mL of concentrated sulfuric acid, and 100 μL of 0.1% (w/v) carbazole reagent (made up in 100% ethanol) were mixed, incubated in a water bath at 60 °C for 1 h, and cooled to room temperature, and absorbance was measured at 530 nm against a water blank. A stock solution of pectin from apples was used at concentrations in the range of 0−200 μg/mL to generate a standard curve. Cell Proliferation Assay. HT-29 cells were cultured in 25 cm2 flasks with Dulbecco’s Modified Eagle’s (DMEM) medium supple-

polyphenols extracted from frozen and freeze-dried blueberry powder. We have also evaluated the ability of these polyphenols to inhibit α-glucosidase, a key enzyme in the human stomach responsible for catabolizing maltose, a disaccharide, that contributes to postprandial increase in blood glucose.

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MATERIALS AND METHODS

Blueberries. Frozen wild blueberries (low-bush) obtained from the local supermarket were divided in two batches: (1) kept as frozen blueberries and (2) used for making blueberry freeze-dried powder. Blueberries were freeze-dried (at −45 ± 5 °C and 100 Pa/1 mbar) in a freeze-dryer (Thermo Savant, USA) for 72 h (2% moisture content on dry basis), milled with a blender, and stored in airtight containers at −20 °C until further analysis. Blueberries from the same lot were used for all experiments. Chemicals and Reagents. All chemicals and solvents were purchased from Fisher Scientific (Ottawa, ON, Canada) or SigmaAldrich (Oakville, ON, Canada). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), L-glutamine, trypsin−EDTA, sulforhodamine B (SRB), acetic acid, and Tris base were purchased from Sigma-Aldrich (Oakville, ON, Canada). Cyanidin- and delphinidin-3-glucosides were purchased from Chromadex (Irvine, CA) and malvidin-3-glucoside from extrasynthese (Genay, Cedex, France). Human epithelial colorectal adenocarcinoma cell line HT-29 and human normal epithelial colon cell line CRL-1790 were obtained from the American Type Culture Collection (Rockville, MD). Minimal Essential Medium Eagle (MEME) was purchased from Hyclone (Thermoscientific, Canada). Phosphate-buffered saline (PBS) 10x, penicillin−streptomycin solution, and trypan blue were from Gibco (Bethesda, MD). Polyphenol Extraction. Methanol and water were used as extraction solvents. Methanolic extraction enabled a more efficient way of extracting phenolic components, and water, on the other hand, simulated the state where anthocyanins are extracted and liberated after consumption as food. One part of frozen blueberries was homogenized with two parts of solvent (50 g of blueberries in 100 mL of solvent) using a shear homogenizer (Brinkman Instruments, Westbury, NY) fitted with a Polytron PTA 10 probe for 5 min. The homogenates were centrifuged (Beckman J2-21, Missisauga, ON) at 27 000g for 20 min; the supernatants were collected and stored in amber tubes at −20 °C for further analysis. Separation of Free and Bound Polyphenols. Blueberry extracts from the aqueous and methanolic crude extracts (10 mL) were transferred into a dialysis membrane reservoir (6−8 kDa cutoff, Spectrapor, Spectrum Laboratories, Houston, TX) previously soaked and rinsed with distilled water. Dialysis was carried out in 500 mL of distilled water at 4 °C for 24 h. The dialyzed (complexed polyphenols) and the dialyzate (free polyphenols) were collected and used for various analyzes.12 Total Polyphenol Content. Total polyphenol contents of the samples were estimated according to the Folin−Ciocalteau method.13 An aliquot of extract was mixed with 50% (v/v) of ethanol in water, to a final volume of 100 μL. Reaction mixture was prepared by adding 500 μL of distilled water, 50 μL of Folin Ciocalteau reagent (2 N), and 100 μL of 5% (w/v) sodium carbonate solution to the sample, vortexed, and incubated in the dark for 1 h. Samples were mixed thoroughly after incubation, and the absorbance was measured at λmax = 725 nm using a Beckman Coulter DU 800 spectrophotometer (Beckman Coulter Inc., IN). A standard curve was generated using gallic acid with concentrations ranging from 0 to 200 μg/mL. Polyphenol concentrations were expressed as weight of gallic acid equivalent per gram fresh weight of fruit. Dry weight of a known amount of polyphenols was determined after freeze-drying. While expressing polyphenol content of blueberries from dry powder, the value was expressed as fresh weight equivalent. Solid-Phase Extraction. Solid-phase extraction (SPE) with SepPak C18 (Waters Corporation, MA) cartridge was used to purify polyphenols.14 The columns were washed with water and polyphenols eluted with 100% methanol. All samples were adjusted to 1 mg/mL of 2936

DOI: 10.1021/acs.jafc.5b00016 J. Agric. Food Chem. 2015, 63, 2935−2946

Article

Journal of Agricultural and Food Chemistry Table 1. Total Polyphenol and Anthocyanin Content of Different Blueberry Extracts polyphenol (mg GAE/g fruit fresh weight)a sample frozen blueberries frozen blueberries blueberry freeze-dried powder blueberry freeze-dried powder a

solvent

crude extract

dialyzed extract

water methanol water methanol

± ± ± ±

± ± ± ±

1.66 3.20 2.00 3.63

a

0.03 0.07b 0.15a 0.19b

0.57 0.97 0.72 0.80

dialyzate

a

0.02 0.01b 0.01a 0.02b

0.79 2.21 1.21 2.82

± ± ± ±

0.02a 0.09b 0.01a 0.04b

The data represent the mean ± SEM from three independent analyses. Means with different superscript letters are statistically significant (p < 0.05).

mented with 10% (v/v) FBS, 200 mM L-glutamine, and 1% (v/v) penicillin−streptomycin. CRL-1790 cells were cultured in Eagle’s minimal essential medium (EMEM) contained in 25 cm2 flasks and supplemented with FBS, penicillin−streptomycin, and L-glutamine as described above. Both cell lines were incubated at 37 °C in a humidified chamber including 5% CO2. Cells were incubated with blueberry extracts for 24 and 48 h. HT-29 cells seeded at 2.0 × 103 cells/well and CRL-1790 seeded at 1.0 × 103 cells/well in 96-well plates were incubated for 24 h to attach to the surface and achieve log phase growth at the time of treatment (time 0). Stock solutions were prepared containing 1 mg/mL of polyphenols dissolved in water and diluted in growth media to obtain concentrations of 10, 25, 50, 75, and 100 μg/mL of media for each sample and immediately tested for antiproliferative activity. Negative control cells were supplied with growth media, and blank wells contained growth media only. The medium was changed every 24 h. Cell proliferation assay was carried out according to a modification of procedure used by Vichai and Kirtikara.20 After 24 and 48 h of treatment, cells were stained with sulforhodamine dye (0.4% dissolved in 1% acetic acid, 50 μL per well), and the optical density of each well was measured at 570 nm using a Synergy HT Multi Detection Microplate Reader (Bio-Tek Instruments, Winooski, VT), and KC4 version 3.03 Power Report Bio-Tek software was used to analyze the data. Statistical Analysis. Statistical analyses were conducted by using Graph Pad Prism software, version 4. Results with multiple means were compared by using one-way analyses of variance (ANOVA), which was followed by Tukey’s test to evaluate the level of significance. Significantly different means (p < 0.05) are denoted by different letters.

Table 2. Major Polyphenols Identified in Blueberry Extracts by HPLC-MS-ESI peaka

tRb (min)

1 2 3 4 5 6 7 8 9 10 11 12 13

15.75 22.00 24.91 26.91 28.70 30.27 33.30 35.05 36.77 39.03 40.56 45.15 47.44

353, 465, 465, 435, 449, 479, 463, 449, 493, 433, 493, 463, 507,

14

49.06

535, 367, 302, 204, 148

15

52.98

435, 303, 229, 153

16

55.54

507, 347, 331, 315, 287, 153

major ionsc

compoundd

336, 288, 180 302, 148 302, 148 302, 132 287,148 316, 148 301, 148 316, 132 331, 148 301, 132 331, 148 331, 132 367, 302, 204, 148

chlorogenic acid delphinidin 3-O-galactoside delphinidin 3-O-glucoside delphinidin 3-O-arabinoside cyanidin 3-O-glucoside petunidin 3-O-galactoside peonidin 3-O-galactoside petunidin 3-O-arabinoside malvidin 3-O- galactoside peonidin 3-O-arabinoside malvidin 3-O-glucoside malvidin 3-O-arabinoside delphinidin 6-acetoyl,3-Oglucoside malvidin 6-acetoyl,3-Oglucoside quercetin 3-O-arabinoside/ xyloside syringetin 3-O-galactoside/ glucoside

a

Peaks were recorded at 260 nm, the absorbance maximum of phenolic moiety in polyphenols. bRetention time for individual component is denoted as tR. cMass spectra for anthocyanins were obtained in the positive ion mode, and those for phenolics and flavonoids in the negative ion mode. dCompounds were identified by comparing the mass spectra to authentic standards that are available at www.metabolomics.jp.

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RESULTS Polyphenol Composition of Blueberry Extracts. Total polyphenol content from different preparations of blueberries ranged from 1.6 to 3.6 mg GAE/g frozen fruit (Table 1). Polyphenol content of methanol extracts of frozen blueberries and freeze-dried blueberries were much higher than that obtained after water extraction, suggesting the presence of significant amounts of bound polyphenols that are not extractable with water. Dialyzed fraction and dialyzate of methanol extracts also possessed increased levels of polyphenols as compared to those from water extracts. Further experiments were largely carried out using polyphenols obtained after water extraction of frozen blueberries, as this was also similar to the levels obtained after water extraction of freeze-dried blueberries. Occasional comparisons have also been made with polyphenols obtained from methanol extracts. Identification and Quantification of Phenolic Compounds. Polyphenols from the original extract (crude extract), dialyzed extract, and dialyzate of blueberry homogenates were analyzed to compare their composition. Major phenolic components identified in the water extracts from frozen blueberries are given in Table 2. These components were also isolated after methanol extraction and dialysis of the extracts which were similar as in water extract but generally higher in amounts (data not shown). Chlorogenic acid was the

major phenolic acid in blueberry extracts. Glucosides, galactosides, and arabinosides of major aglycones including delphinidin, cyanidin, petunidin, peonidin, and malvidin were observed. Glycosylated flavonoids such as quercetin arabinoside (xyloside) and syringetin galactoside (glucoside) were also identified. Acetoylated anthocyanins of delphinidin, and malvidin glucosides, were major components among anthocyanins. HPLC profiles of polyphenols purified from water extracts, dialyzed extract, and the dialyzate of frozen blueberries revealed the presence of 16 major phenolic compounds (Figure 1). The content of individual phenolic/flavonoid components and anthocyanins in these samples is shown in Table 3 and Table 4, respectively. Chlorogenic acid was the major phenolic acid component in blueberries, approaching nearly 50% of the total phenolic components in the aqueous crude extract and the dialyzate. Quercetin arabinoside (xyloside) was the major flavonoid glucoside, present in both crude extract and dialyzate. In addition, flavonoids such as myricetin, quercetin arabinoside, and syringetin galactoside (glucoside) were also present in aqueous crude extracts. Several other phenolic components such as isorhamnetin, 4-O-feruloylquinic acid, and myricetin 2937


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Figure 1. HPLC profile of blueberry polyphenols, detected at 260 nm. (a) Crude extract, (b) dialyzed fraction, (c) dialyzate fraction. Polyphenols from berries were extracted with water and subjected to purification using sep-pak C18 columns before analysis. Identification of the peaks denoted by numbers is given in Table 2

calculated from the linear phase of the activity curve (Table 5). Maximal levels of DPPH radical scavenging and hydroxyl radical scavenging were observed in the free polyphenol fraction obtained after dialysis of frozen blueberry extracts, and these values were nearly twice as high as in the crude extract. The highest level of superoxide scavenging was observed in the dialyzed fraction (polyphenols bound to the matrix) of frozen blueberry extracts. Dialyzate of the frozen blueberry extract was as efficient as the standards in DPPH scavenging and far reduced in hydroxyl radical scavenging. For freeze-dried blueberries, dialyzed extract showed the highest DPPH scavenging and superoxide scavenging activities. But the dialyzate of the freeze-dried berries showed the maximum level of hydroxyl radical activity (Table 5). α-Glucosidase Inhibition. Polyphenols isolated from water and methanol extracts of blueberries (crude extracts, dialyzed extract, and dialyzate) from both frozen fruits and freeze-dried powder were evaluated for their potential to inhibit α-glucosidase, a key sugar catabolizing enzyme in the stomach responsible for the liberation of sugars from disaccharides such as maltose, produced during the digestion of starch. As shown in Figure 2, the specific inhibition of α-glucosidase by the crude extract, the dialyzed extract, and dialyzate was highly variable, with the dialyzed extracts showing the most efficient inhibition and the dialyzate showing the least inhibition. The crude extracts of polyphenols in methanol showed levels of glucosidase inhibition higher than those of water extracts from both frozen and freeze-dried blueberries. A similar pattern was observed while using the dialyzed extract, but with effectiveness nearly twice as high as that observed in the crude extracts with maximal inhibition observed between 80% and 100%. Interestingly, the dialyzed methanol extract was

Table 3. Flavonoid and Phenolic Acid Composition (mg/g Fresh Weight) of Crude Extract, Dialyzed Extract, and Dialyzate Fraction Obtained from Frozen Blueberries Extracted with Watera phenolic component isorhamnetin myricetin chlorogenic acid 4-O-feruloylquinic acid quercetin- 3-Oarabinoside/ xyloside syringetin 3-Ogalactoside/ glucoside total

crude extract

dialyzed extract

dialyzate

± ± ± ±

Trb 0.009 ± 0.000b 0.031 ± 0.014b 0.062 ± 0.006b

Tr 0.010 ± 0.002b 0.190 ± 0.026a Tr

0.091 ± 0.021a

0.036 ± 0.006b,c

0.054 ± 0.005c

0.020 ± 0.006a

Tr

0.050 ± 0.004b

0.420 ± 0.040a

0.128 ± 0.015b

0.304 ± 0.037c

0.028 0.024 0.216 0.041

0.003a 0.001a 0.010a 0.008a

The values given are mean ± SEM from three independent analyzes. Means with different superscript letters in the same row are statistically significant (p < 0.05). bTr = traces. a

were detected in very low amounts (mass spectral identification). Aqueous dialyzed fraction contained less than 10% of the polyphenols present in the original crude extract, having most of the components typically present in the crude extract and the dialyzate. Antioxidant Activity. Antioxidant activities of free and complexed blueberry polyphenols isolated from frozen- and freeze-dried blueberries were evaluated to observe if freezedrying affected complex formation with the food matrix, and this in turn affected the antioxidant activity of polyphenols. The specific quenching values per 10 μg polyphenols were 2938


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Journal of Agricultural and Food Chemistry Table 4. Composition of Anthocyanins (mg/g Fresh Weight) of Crude Extract, Dialyzed Extract and Dialyzate Fraction Obtained from Frozen Blueberry Fruits Extracted with Watera anthocyanin component delphinidin 3-Ogalactoside delphinidin 3-Oglucoside delphinidin 3-Oarabinoside cyanidin 3-Oglucoside petunidin 3-Ogalactoside peonidin 3-Ogalactoside petunidin 3-Oarabinoside malvinidin 3-Ogalactoside peonidin 3-Oarabinoside malvidin 3-Oglucoside malvidin 3-Oarabinoside delphinidin 6acetoyl-3-Oglucoside malvidin 6-acetoyl-3O-glucoside total

crude extract

dialyzed extract

dialyzate

0.007 ± 0.001a

Trb

0.009 ± 0.001a

0.015 ± 0.003a

Tr

0.011 ± 0.003a

Tr

Tr

Tr

0.016 ± 0.003a

Tr

Tr

0.012 ± 0.003a,b

Tr

0.014 ± 0.003b

0.019 ± 0.004a

Tr

0.010 ± 0.003b

Tr

Tr

0.055 ± 0.005a

0.077 ± 0.011a

Tr

0.061 ± 0.012a,b

Tr

Tr

0.017 ± 0.005a

0.072 ± 0.007a

Tr

Tr

0.024 ± 0.004a

Tr

Tr

0.095 ± 0.014a

0.029 ± 0.008b

0.101 ± 0.022a

0.116 ± 0.010a

0.054 ± 0.018b,c 0.101 ± 0.036c

0.453 ± 0.056a

0.083 ± 0.025b

0.379 ± 0.089a

The values given are mean ± SEM from three independent analyzes. Means with different superscript letters in the same row are statistically significant (p < 0.05). bTr = traces. a

Table 5. In Vitro Antioxidant Activities of Crude Water Extract, Dialyzed Extract, and Dialyzate from Frozen and Freeze-Dried Wild Blueberriesa specific scavenging (% reduction per 10 μg of polyphenols) samples crude extract dialyzed extract dialyzate vitamin C trolox crude extract dialyzed extract dialyzate vitamin C trolox

DPPH radical

superoxide radical

Frozen Blueberries 17.61 ± 0.56b 30.78 ± 9.17a 24.66 ± 2.10b 46.06 ± 2.49a 33.12 ± 4.30aA 7.80 ± 1.99bA 40.90 ± 3.23 21.62 ± 1.57 33.57 ± 2.60 22.18 ± 2.90 Freeze-Dried Blueberries 21.73 ± 0.96a 23.11 ± 5.62a a 22.63 ± 6.38 35.55 ± 8.0a aB 11.40 ± 3.80 28.84 ± 6.99aB 40.90 ± 3.23 21.62 ± 1.57 33.57 ± 2.60 22.18 ± 2.90

Figure 2. Percent inhibition of α-glucosidase activity of polyphenols isolated from blueberry extract, dialyzed extract, and dialyzate fractions. Frozen blueberries and freeze-dried blueberries were extracted with water or methanol, and the polyphenols were subjected to dialysis against water and purified using sep-pak C18 columns. Inhibition of α-glucosidase activity was performed in the presence of increasing levels of polyphenols. The activity curves are denoted as follows: dark green square, frozen blueberries extracted with water; purple triangle, frozen blueberries extracted with methanol; light green triangle, freeze-dried powder extracted with water; blue diamond, freeze-dried powder extracted with methanol. Catechin was used as a standard: black circle. The results shown are mean ± SEM from three independent experiments.

hydroxyl radical 11.17 ± 3.48b 13.53 ± 4.44b 25.90 ± 2.60aA − 79.30 ± 1.34 11.54 ± 3.20b 6.85 ± 2.03b 40.43 ± 1.26aB − 79.30 ± 1.34

shown) nor catechin inhibited α-glucosidase activity more than 20% at 100 μg/mL. Antiproliferative Activity. Colon cancer (HT-29) and normal colon (CRL-1790) cells were incubated in the presence or absence of free and complexed blueberry polyphenols (dialyzed and dialyzate fractions) at concentrations ranging from 0 to 100 μg total polyphenols/mL medium, and proliferation was evaluated using the SRB binding assay relative to an untreated control (only medium without polyphenols) (Figure 3). Results clearly indicated that all samples exhibited significant inhibitory activities on the growth of both HT-29

a

Data are representative of 3 independent assays, and expressed as mean ± SEM. Means with different superscript letters in the same column are statistically significant (p < 0.05).

twice as effective as the dialyzed water extract from frozen blueberries at 20 μg/mL level. The dialyzate from extracts of both frozen and freeze-dried blueberries comprising free polyphenols showed the least amount of α-glucosidase inhibition (50 μg/mL). Methanolextracted and purified polyphenols, dialyzed extract, and dialyzate from frozen blueberries showed a similar degree of antiproliferative action on HT-29 cells as compared to similar extracts from freeze-dried blueberries (Figure 3). Inhibitory properties of these extracts were slightly reduced in CRL 1790 2940


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lower than what was observed in the dialyzed extract of blueberries (Table 1). Antioxidant Activity in Mixed Complexes. The ability of fruit matrix (i.e., pectin, cellulose, and pectin + cellulose) to scavenge DPPH radicals was assessed in comparison to the dialyzed and dialyzate fractions obtained from blueberry homogenate. Fruit matrix alone (obtained after the extraction of the polyphenols) was tested after mixing with equivalent levels of polyphenols that were present in the original blueberry homogenate. Blueberry dialyzed extract containing polyphenols showed a dose-dependent increase in DPPH scavenging ability (Figure 5). Dialyzate from blueberry extract containing free polyphenols showed almost 90% scavenging of DPPH radicals at the highest concentration tested, followed by complexed polyphenols obtained from dialyzed extract of blueberries. Natural food matrix isolated from ethanol-bleached blueberries before and after dialysis showed a very low scavenging ability of DPPH at ∼10%. Different solutions containing food matrixes made of pectin or cellulose showed ∼5% inhibition. When complexed as in the dialyzed extract, the matrix tends to reduce the antioxidant capacity of polyphenols in the range of 10−20% (Figure 5). Antiproliferative Activity in Mixed Complexes. Antiproliferative activities of blueberry polyphenols and different food matrixes were evaluated using colon cancer cell line HT29. As shown in Figure 6, free polyphenols at the highest concentration (100 μg/mL) inhibited approximately 80% of the growth of HT-29 cells. By contrast, when the same amount of polyphenols was naturally complexed with the blueberry fruit matrix, there was only 50% inhibition. Samples containing 100 μg/mL of the fruit matrix, or the dialyzed food matrix, showed only 10% and 30% inhibition of cell growth after 24 and 48 h incubation periods, respectively. In another experiment, when the dialyzate containing free polyphenols or purified polyphenols was mixed with pectin, cellulose, or a mixture of pectin and cellulose and incubated with HT 29 cells, antiproliferative action was nearly similar for 24 h incubation, but during prolonged incubation for 48 h, polyphenol−pectin mixture tended to show a higher degree of antiproliferative activity, suggesting that during long-term incubation in media, pectin may complex with proteins, resulting in enhanced bioavailability of polyphenols and an increase in antiproliferative activity (Figure 7). These aspects need further investigation.

blueberries and freeze-dried blueberries, as well as their dialyzed extracts and dialyzates, showed a relatively higher antiproliferative activity against CRL 1790 cells at concentration ranges lower than 50 μg/mL. Pectin Content of Blueberry Extracts. To further understand the potential nature of polyphenol food matrix interactions, levels of pectin, the major cell wall component in ripe fruit, were analyzed in different berry fractions (Figure 4).

Figure 4. Pectin contents of different blueberry food matrixes. A set of blueberry fruit (50 g) was bleached with ethanol to remove the polyphenols completely, and the resulting fruit were subjected to homogenization in water, removal of debris by centrifugation, and dialysis. These fractions are denoted as food matrix and dialyzed food matrix. A separate set (50 g) was subjected to water extraction, removal of debris, centrifugation, and dialysis. Pectin content was estimated by a colorimetric method. The data are from three separate analyses and expressed as mean ± SEM.

There were no significant differences in pectin content between crude extracts and dialyzed extracts of decolorized fruits and the original blueberry extracts and dialyzed extracts. The average pectin content in all samples was approximately 1.5 mg/g frozen weight of blueberries. To evaluate the ability of purified carbohydrate-rich matrixes to complex with polyphenols, varying concentrations of pectin and cellulose were mixed with 1 mg gallic acid equivalent of free polyphenols. To confirm that these polyphenols could form complexes with pectin and cellulose after mixing, the mixtures were dialyzed overnight. Table 6 shows the proportion of polyphenols complexed with pectin−cellulose and those that are free and recovered in the dialyzate fraction after dialysis. A fixed amount of polyphenols (1.3 mg) was mixed with different solutions: 0.5% w/v pectin, 0.5% w/v cellulose, and 0.5% w/v pectin−cellulose, and total polyphenols were quantified. During dialysis, over 90% of the polyphenols leaked out of the bag when polyphenols were alone or mixed with cellulose. When mixed with pectin or pectin and cellulose, there was some retention of the polyphenols within the dialysis bag, indicating a weak binding between pectin and polyphenols, but this was far

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DISCUSSION Public interest in blueberries as a functional food has increased in recent years because of its health benefits. Blueberry is commonly consumed fresh, frozen, and as a powder. Simple and acylated anthocyanins have been found in some low bush cultivars.21,22 Overall, the profile of polyphenols in this study is consistent with those reported previously in the literatur-

Table 6. Effect of Dialysis on the Retention of Polyphenols by Pectin (0.5%), Cellulose (0.5%), and a Mixture of Pectin and Cellulose (0.25% each)a original extract sample polyphenols alone pectin−polyphenol cellulose−polyphenol pectin−cellulose−polyphenol a

dialyzed extract

phenolic content (mg)

recovery (%)

± ± ± ±

100 100 100 100

1.3 1.3 1.3 1.3

0.05 0.03 0.06 0.04

dialyzate


recovery (%)

± ± ± ±

7.7 7.7 7.7 6.2

0.10 0.10 0.10 0.08

0.003 0.002 0.006 0.001


recovery (%)

± ± ± ±

92 86 91 89

1.20 1.13 1.18 1.16

0.02 0.09 0.18 0.02

Data presented are mean ± SEM from three separate analyzes. The values in columns are not significantly different from one another at P < 0.5. 2941


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Figure 5. A comparison of DPPH radical scavenging activities of isolated bleached blueberry fruit matrix, pectin, and cellulose (each at 100 μg/mL) alone, with the dialyzed extract (complexed polyphenols) and with the dialyzate (free polyphenols) of water extracted blueberries (top panel). The concentrations indicated (x-axis) are for both polyphenols (as estimated by Folin’s reagent) and the matrix (weight). The bottom panel shows the effect of pectin and a combination of cellulose and pectin (100 μg/mL) on the DPPH-radical scavenging activity of free polyphenols isolated from blueberries. The data represent mean ± SEM from three separate experiments.

Figure 6. Effect of food matrix (100 μg/mL) isolated from blueberries on antiproliferative activity of free polyphenols (dialyzate) and complexed polyphenols (dialyzed extract) monitored at 24 h after treatment (top panel) and 48 h after treatment (bottom panel) using HT 29 colorectal cancer cells. The data are mean ± SEM from three independent experiments.

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Figure 7. Antiproliferative activity of free polyphenols (dialyzate) in combination with various mixtures of polyphenols, pectin, and cellulose (100 μg/mL) evaluated using HT 29 cells. Antiproliferative activity was monitored at 24 h (top panel) and 48 h (bottom panel). The data are mean ± SEM from three independent experiments.

e.23−26Organic acids are also helpful in the stabilization of ascorbic acid and anthocyanins in frozen and processed berries.27 Blueberries are rich in phenolic acids such as benzoic acid and cinnamic acid derivatives. Benzoic acid derivatives include vanillic acid, syringic acid, gallic acid, protocatechuic acid, mhydroxybenzoic acid, p-hydroxybenzoic acid and ellagic acid. Major cinnamic acid derivatives include chlorogenic acid, caffeic acid, ferulic acid, quinic acid and coumaric acid.23 Polyphenols in blueberries are naturally esterified with other phenolic acids or sugars, suggesting that they are frequently found as glucosides such as quercetin glucoside, myricetin glucoside, and kaempferol glucoside.28 In our study, chlorogenic acid was the major component identified, and other phenolic acids were nondetectable or occurred as minor components eluting before chlorogenic acid. 10 Flavonoid derivatives of quercetin, rhamnetin, syringetin, and myricetin were also identified. In blueberry skin and flesh, delphinidin, cyanidin, petunidin, peonidin, and malvidin monoglycosides (glycosides, galactosides and arabinosides) are the main anthocyanins. Because of the diversity in the glycosylation and acylation pattern, more than 25 anthocyanins have been identified in blueberries. The distribution of anthocyanins in five different blueberry genotypes have been reported,29 where delphinidin was the most abundant anthocyanin (27−40%), followed by malvidin (22−33%), petunidin (19−26%), cyanidin (6−14%), and peonidin (1−5%). Nearly equal distribution of anthocyanins was observed in blueberry fruits analyzed in this study.

Anthocyanins are rarely ingested on their own but as a component of fruits or vegetables or in meals containing other simple and macromolecular food components. Studies carried out by Mazza and co-workers30 have shown that there are significant differences in the absorption rates of anthocyanins when consumed either alone or with high fat meals.30,31 In a study32 on red grape juice, a synergistic enhancement in intestinal anthocyanin absorption by glucose was observed, as compared to that from red wine. It has also been shown that isolated polyphenols from grape juice are more effective in arresting the growth of tumor in mice than those isolated from red wine, when provided through gavage.33 Although it is generally believed that the food matrix may not affect the functionality of polyphenols, molecular interactions between polyphenols and other components present in the food matrix ultimately may alter their antioxidant function and bioefficacy.12,34,35 For instance, 34−40% of polyphenols in red wine seem to be complexed to dietary fiber.35 In another study, nearly 25% of polyphenols present in grape juice concentrate were found to be complexed with carbohydrates.12 Thus, foodmatrix interactions may play a major role in the functionality of polyphenols. In this study, we analyzed the potential modulatory role of food matrix in polyphenol function. During ripening, the structural matrixes such as cellulose and pectin undergo degradation. As the compartmentalization of ripening tissue decreases, the polyphenols that are normally confined to vacuoles may leak out and form complexes with cellular 2943


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antiproliferative activities, the dialyzed extract containing complexed polyphenols showed the highest inhibitory activity toward α-glucosidase. It has been previously hypothesized41 that the inhibitory effectiveness of berry fruit extracts against αglucosidase was related to their anthocyanin content. The results from this study suggest that the complexed polyphenols or their interactions with other components of the food matrix enhance their ability to inhibit α-glucosidase activity. In vivo studies with healthy human volunteers demonstrated that freeze-dried wild blueberry powder increased the serum antioxidant status.42 Whole blueberry freeze-dried powder also reduced the insulin resistance in adipose tissue and increased the function of β cells in male C57BL mice fed high fat diets, suggesting that processed fruits are equally effective in providing health benefits.42,43 Thus, polyphenols, whether free or complexed with fruit matrixes, can thus provide multiple protective roles in the human body. Apart from the phenolic components, flavonoids, and anthocyanins, blueberries are a rich source of procyanidins which are dimers, trimers, and polymers of flavan-3-ols, catechin, and epicatechin.44 These components have not been analyzed in the present study. Because of their complex structure, procyanidins are ideally suited to form molecular complexes with pectin and other cell wall polymers. It is also highly likely that the health benefits observed in blueberries may in part be due to their procyanidin content.44 In conclusion, interactions between polyphenols and food matrix can play an important role in the bioactivity of blueberries. Although complex formation did not reduce the antioxidant and α-glucosidase activities of blueberry polyphenols, it slightly reduced the antiproliferative activity. These results suggest that different fractions of polyphenols obtained in frozen or freeze-dried blueberry products may provide multiple effects such as antioxidant activity, regulation of glucose homeostasis, and antiproliferative functions to different extents depending on their complexed or free state. The presence of a food matrix may positively affect the stability of polyphenols in the gastrointestinal tract, as well as the extent of absorption and their metabolism in vivo. A better understanding of which compounds form complexes with the polyphenols is needed, as well as their stability in the gastrointestinal tract to obtain relevant information about the bioavailability and metabolism of phenolic compounds in blueberries.

macromolecules. Thus, it was interesting to see if food matrix isolated from ripe fruits or those reconstituted by components such as cellulose and pectin had any influence in polyphenol functionality, such as its antioxidant function or anticancer properties. These interactions may have an impact on the delivery and absorption of polyphenols in vivo. Our results show that nearly 28% of the polyphenols in blueberry crude extracts are naturally complexed with the food matrix. These results are in agreement with previous studies12 demonstrating that approximately 25% of nondialyzable polyphenols in grapes were bound to carbohydrates and lipids. The present study suggests that food matrix alone, as well as the synthesized food matrixes used in the experiments, did not have a considerable antioxidant activity. When free polyphenols were mixed with different concentrations of pectin or/and cellulose, their scavenging capacity of the DPPH radical was similar to that of free polyphenols. As well, isolated matrix devoid of polyphenols by itself showed minimal antioxidant or antiproliferative activity. When polyphenols are mixed with high amounts of pectin or cellulose, antioxidant activity of polyphenols does not decrease. However, when the dialyzed extract containing naturally complexed polyphenols was mixed with food matrix, it showed a lower antioxidant activity and antiproliferative activity as compared to that of free polyphenols. This may be due to complex formations involving key hydroxyl groups of the polyphenols with polygalacturonic acid moieties of pectin that make polyphenols less amenable to scavenge free radicals. Despite the fact that there are numerous reports of inhibition of cancer cell growth and proliferation in vitro by purified anthocyanin-rich extracts or crude extracts, very few studies have been conducted to delineate potential influences of food matrix on the biological activity of components studied. In this study, the antiproliferative action of free polyphenols, complexed polyphenols, and food matrix was compared separately.36,37 It is of great interest to understand the effect of the food matrix on the biological activity of polyphenols to develop processing techniques that take advantage of such interactions and lead to the production of high quality and value-added products. Although, blueberry food matrix did not show a considerable effect on antiproliferative activity, there was a significant decrease in the inhibitory activity when polyphenols were naturally complexed with the blueberry food matrix. By contrast, various mixtures containing free polyphenols and the synthetic food matrixes did not have the same effect as polyphenols complexed with matrix during extraction. These results suggest that strong interactions between polyphenols and fruit matrixes may affect the bioavailability and efficacy of polyphenols in several ways. Besides having high in vitro antioxidant activity, different fruit extracts have been studied for their ability to inhibit αglucosidase, a key enzyme involved in the digestion of starch into sugars. Inhibition of the activity of carbohydrate-hydrolyzing enzymes has the potential to lower sugar absorption and consequently may provide an additional tool for the management of blood sugar levels by preventing postprandial hyperglycemia, thus controlling the onset of type II diabetes.38,39 Efficacy of the polyphenols in binding with proteins and subsequent enzymatic inhibition could be attributed to the fact that polyphenols are multidentate ligands, capable of binding simultaneously (via different hydroxyl groups) to more than one site of the protein (enzyme).40 In contrast to the reduction observed in antioxidant and

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AUTHOR INFORMATION

Corresponding Author

*Phone: 519-824-4120 x54856. E-mail: [email protected]. Funding

We are grateful to the Ontario Ministry of Agriculture, Food and Rural Affairs for the financial assistance to conduct this research. We also thank CONACYT, Mexico, for providing a scholarship to Julieta C. Betanzo. Notes

The authors declare no competing financial interest.

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REFERENCES

(1) Strik, B. Berry crops: Worldwide area and production systems. In Berry fruit: Value-added products for health promotion; Zhao, Y., Ed.; CRC Press: Boca Raton, FL, 2007; pp 3 − 50. (2) Papadopoulou, A.; Frazier, R. A. Characterization of proteinpolyphenol interactions. Trends Food Sci. Technol. 2004, 15, 186−190.

2944


Article


(23) Su, Z. Anthocyanins and flavonoids of Vaccinium L. Pharm. Crops 2012, 3, 7−37. (24) Sellappan, S.; Akoh, C. C.; Krewer, G. Phenolic compounds and antioxidant capacity of Georgia-grown blueberries and blackberries. J. Agric. Food Chem. 2002, 50, 2432−2438. (25) You, Q.; Wang, B.; Chen, F.; Huang, Z.; Wang, X.; Luo, P. G. Comparison of anthocyanins and phenolics in organically and conventionally grown blueberries in selected cultivars. Food Chem. 2011, 125, 201−208. (26) Wang, S. Y.; Chen, H.; Camp, M. J.; Ehlenfeldt, M. K. Flavonoid constituents and their contribution to antioxidant activity in cultivars and hybrids of rabbiteye blueberry (Vaccinium ashei Reade). Food Chem. 2012, 132, 855−864. (27) Viljakainen, S.; Visti, A.; Laakso, S. Concentrations of organic acids and soluble sugars in juices from Nordic berries. Acta Agric. Scand. 2002, 52, 101−109. (28) Häkkinen, S. H.; Heinonen, I. M.; Karenlampi, S. O.; Mykkanen, H.M.; Ruus-Kanen, J.; Torronen, A. R. Screening of selected flavonoids and phenolic acids in 19 berries. J. Food Res. Int. 1999, 32, 345−353. (29) Cho, M. J.; Howard, L.; Prior, R.; Clark, J. Flavonoid glycosides and antioxidant capacity of various blackberry, blueberry and red grape genotypes determined by high-performance liquid chromatography/ mass spectrometry. J. Sci. Food Agric. 2004, 84, 1771−1782. (30) Mazza, G.; Kay, C. D.; Cottrell, T.; Holub, B. J. Absorption of anthocyanins from blueberries and serum antioxidant status in human subjects. J. Agric. Food Chem. 2002, 50, 7731−7737. (31) Kay, C. D.; Mazza, G.; Holub, B. J.; Wang, J. Anthocyanin metabolites in human urine and serum. Br. J. Nutr. 2004, 91, 933−942. (32) Bitsch, R.; Netzel, M.; Frank, T.; Strass, G.; Bitsch, I. Bioavailability and biokinetics of anthocyanins from red grape juice and red wine. J. Biomed. Biotechnol. 2004, 5, 293−298. (33) Hakimuddin, F.; Tiwari, K.; Paliyath, G.; Meckling, K. Grape and wine polyphenols down-regulate the expression of signal transduction genes and inhibit the growth of estrogen receptornegative MDA-MB231 tumors in nu/nu mouse xenografts. Nutr. Res. (N. Y., NY, U. S.) 2008, 28, 702−713. (34) Rice-Evans, C. A.; Miller, N. J. Antioxidant activities of flavonoids as bioactive components of food. Biochem. Biochem. Soc. Trans. 1996, 24, 790−795. (35) Saura-Calixto, F.; Serrano, J.; Goñi, I. Intake and bioaccessibility of total polyphenols in a whole diet. Food Chem. 2007, 101, 492−501. (36) Seeram, N. P.; Adams, L. S.; Zhang, Y.; Lee, R.; Sand, D.; Scheuller, H. S.; Heber, D. Blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberry extracts inhibit growth and stimulate apoptosis of human cancer cells in vitro. J. Agric. Food Chem. 2006, 54, 9329−9339. (37) Zhao, C.; Giusti, M. M.; Malik, M.; Moyer, M. P.; Magnuson, B. Effects of commercial anthocyanin-rich extracts on colonic cancer and nontumorigenic colonic cell growth. J. Agric. Food Chem. 2004, 52, 6122−6128. (38) Shetty, K. Biosynthesis of rosmarinic acid and applications in medicine. J. Herbs Spices Med. Plants 2001, 8, 161−181. (39) Grace, M. H.; Ribnicky, D. M.; Kuhn, P.; Poulev, A.; Logendra, S.; Yousef, G. G.; Raskin, I.; Lila, M. A. Hypoglycemic activity of a novel anthocyanin-rich formulation from lowbush blueberry, Vaccinium angustifolium Aiton. Phytomedicine 2009, 16, 406−415. (40) Freitas, V.; Carvalho, E.; Mateus, N. Study of carbohydrate influence on protein-tannin aggregation by nephelometry. Food Chem. 2003, 81, 503−509. (41) Kay, C. D.; Holub, B. J. The effect of wild blueberry (Vaccinium angustifolium) consumption on postprandial serum antioxidant status in human subjects. Br. J. Nutr. 2002, 88, 389−397. (42) DeFuria, J.; Bennett, G.; Strissel, K. J.; Perfield, J. W.; Milbury, P. E.; Greenberg, A. S.; Obin, M. S. Dietary blueberry attenuates whole-body insulin resistance in high fat-fed mice by reducing adipocyte death and its inflammatory sequel. J. Nutr. 2009, 139, 1510−1516.

(3) Williamson, G.; Manach, C. Bioavailability and bioefficacy of polyphenols in humans. II. Review of 93 intervention studies. Am. J. Clin. Nutr. 2005, 81, 243S−255S. (4) Wang, L. S.; Stoner, G. D. Anthocyanins and their role in cancer prevention. Cancer Lett. 2008, 269, 281−290. (5) Jacob, J. K.; Tiwari, K.; Correa-Betanzo, J.; Misran, A.; Chandrasekaran, R.; Paliyath, G. Biochemical basis for functional ingredient design from fruits. Annu. Rev. Food Sci. Technol. 2012, 3, 79−104. (6) Prior, R. L.; Martin, A.; Cao, G.; Sofic, E.; McEwan, J.; O’Brien, C.; Lischner, N.; Ehlenfeldt, M.; Krewer, G.; Mainland, C. M. Antioxidant capacity as influenced by total phenolics and anthocyanin content, maturity and variety of Vaccinium species. J. Agric. Food Chem. 1998, 46, 2686−2693. (7) Kyle, J. A. M.; Duthie, G. G. Flavonoids in foods. In Flavonoids: Chemistry, Biochemistry and Applications; Andersen, O. M., Markham, K. R., Eds.; CRC Press, Taylor and Francis: Boca Raton, FL, 2006; pp 219−261. (8) Andersen, O. M.; Jordheim, M. The anthocyanins. In Flavonoids: Chemistry, Biochemistry and Applications; Andersen, O. M., Markham, K. R., Eds.; CRC Press, Taylor and Francis: Boca Raton, FL, 2006; pp 471−551. (9) Turgeon, S. L.; Rioux, L. E. Food matrix impact on macronutrients nutritional properties. Food Hydrocolloids 2011, 25, 1915−1924. (10) Correa-Betanzo, J.; Allen-Vercoe, E.; McDonald, J.; Schroeter, K.; Corredig, M.; Paliyath, G. Stability and biological activity of wild blueberry (Vaccinium angustifolium) polyphenols during simulated in vitro gastrointestinal digestion. Food Chem. 2014, 165, 522−531. (11) Aguilera, J. M.; Stanley, D. W.; Baker, K. W. New dimensions in microstructure of food products. Trends Food Sci. Technol. 2000, 11, 3−9. (12) Jacob, J. K.; Paliyath, G. Physicochemical characteristics of nanovesicle−carbohydrate complexes in grape juice concentrate. J. Agric. Food Chem. 2008, 56, 1305−1315. (13) 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. Methods Enzymol. 1999, 299, 52− 57. (14) Jacob, J. K.; Hakimudin, F.; Paliyath, G.; Fischer, H. Antioxidant and antiproliferative activity of polyphenols in novel high-polyphenol grape lines. Food Res. Int. 2008, 41, 419−428. (15) Taylor, K. A. C. C. A colorimetric method for the quantitation of galacturonic acid. Appl. Biochem. Biotechnol. 1993, 43, 51−54. (16) Molyneux, P. The use of stable free radical diphenyl picrylhydrazyl (DPPH) for estimating antioxidant activity. J. Sci. Technol. 2004, 26, 211−219. (17) Madamanchi, N. R.; Donahue, J. L.; Cramer, C. L.; Alscher, R. G.; Pedersen, K. Differential response of Cu, Zn superoxide dismutase in two pea cultivars during a short-term exposure to sulfur dioxide. Plant Mol. Biol. 1994, 26, 95−103. (18) Halliwell, B.; Gutteridge, J. M. C.; Aruoma, O. I. The deoxyribose method: A simple “test-tube” assay for determination of rate constants for reactions of hydroxyl radicals. Anal. Biochem. 1987, 165, 215−219. (19) Kwon, Y.; Apostolidis, E.; Shetty, K. 2008. Inhibitory potential of wine and tea against α-amylase and α-glucosidase for management of hyperglycemia linked to type 2 diabetes. J. Food Biochem. 2008, 32, 15−31. (20) Vichai, V.; Kirtikara, K. Sulforhodamine B colorimetric assay for antiproliferative activity screening. Nat. Protocols 2006, 1, 1112−1117. (21) Gao, L.; Mazza, G. Quantitation and distribution of simple and acylated anthocyanins and other phenolics in blueberries. J. Food Sci. 1994, 59, 1057−1059. (22) Wu, X.; Prior, R. L. Systemic identification and characterization of anthocyanins by HPLC-ESI-MS/MS in common foods in the United States: Fruits and berries. J. Agric. Food Chem. 2005, 53, 2589− 2599. 2945


Article

Journal of Agricultural and Food Chemistry (43) Prior, R. L.; Wilkes, S. E.; Rogers, T. R.; Khanal, R. C.; Wu, X.; Howard, L. R. Purified blueberry anthocyanins and blueberry juice alter development of obesity in mice fed an obesogenic high-fat diet. J. Agric. Food Chem. 2010, 58, 3970−3976. (44) Prior, R. L.; Lazarus, S. A.; Cao, G.; Muccitelli, H.; Hammerstone, J. F. Identification of procyanidins and anthocyanins in blueberries and cranberries (Vaccinium Spp.) using high-performance liquid chromatography/mass spectrometry. J. Agr. Food Chem. 2001, 49, 1270−1276.

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Complex Formation of Blueberry (Vaccinium angustifolium

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