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Development of a Cranberry Standard for Quantification of Insoluble Cranberry (Vaccinium macrocarpon Ait.) Proanthocyanidins Erica R. Gullickson,† Christian G. Krueger,*,†,‡ Andrew Birmingham,‡ Michael Maranan,‡ and Jess D. Reed†,‡

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Reed Research Group, Department of Animal Sciences, University of WisconsinMadison, 1675 Observatory Drive, Madison, Wisconsin 53706, United States ‡ Complete Phytochemical Solutions LLC, 317 South Street, Cambridge, Wisconsin 53523, United States ABSTRACT: Cranberry proanthocyanidins (PACs) can be partitioned into soluble PACs, which are extracted with solvents, and insoluble PACs, which remain associated with fibers and proteins after extraction. Most research on cranberry products only quantifies soluble PACs because proper standards for quantifying insoluble PACs are lacking. In this study, we evaluated the ability of a cranberry PAC (c-PAC) standard, reflective of the structural heterogeneity of PACs found in cranberry fruit, to quantify insoluble PACs by the butanol−hydrochloric acid (BuOH−HCl) method. For the first time, a c-PAC standard enabled conversion of BuOH−HCl absorbance values (550 nm) to a weight (milligram) basis, allowing for quantification of insoluble PACs in cranberries. The use of the c-PAC reference standard for sequential analysis of soluble PACs by the method of 4(dimethylamino)cinnamaldehyde and insoluble PACs by the method of BuOH−HCl provides analytical tools for the standardization of cranberry-based ingredients. KEYWORDS: cranberry, insoluble proanthocyanidin, butanol−HCl

1. INTRODUCTION Cranberry proanthocyanidin (PAC) oligomers are predominantly made up of epicatechin extender units. The stereochemistry of the flavanol monomers are predominantly 2,3-cistype, with a small proportion of 2,3-trans units.1 Some studies also report the presence of epigallocatechin and catechin units in PAC oligomers.1−4 There are two common series of PAC dimers. The “B-type” series are dimers linked either in the C4−C6 or C4−C8 position, whereas the “A-type” series are dimers linked in the C4−C8 position with an additional C2− O−C7 ether linkage. Cranberry PAC oligomers with a degree of polymerization greater than 2 may incorporate both “Atype” and “B-type” interflavan linkages. By extension of this definition and for purposes of discussing differences among oligomers, PACs that contain one or more “A-type” interflavan bonds in their structure are referred to as “A-type” PACs, whereas PAC oligomers that contain only “B-type” interflavan bonds are referred to as “B-type” PACs.5 Until now, only soluble PACs have been reported to occur in cranberry fruits, sweetened-dried cranberries, cranberry juices, cranberry press cake, cranberry juice concentrates, and powders derived from cranberry juice concentrates. Soluble PACs can be quantified by the method of 4-(dimethylamino)cinnamaldehyde (DMAC)6−8 using a cranberry proanthocyanidin (c-PAC) reference standard that is representative of the structural heterogeneity of PACs found in cranberry fruit and is therefore an accurate standard for the quantification of cranberry PACs by colorimetric methods, such as DMAC and BuOH−HCl methods.7,8 The DMAC method is preferred over the BuOH−HCl method for measuring soluble cranberry PACs because the DMAC method is more specific for flavan-3© XXXX American Chemical Society

ols and does not suffer from interference from existing anthocyanidins in the sample.9 Therefore, the DMAC method was used, as opposed to the BuOH−HCl method, to quantify soluble PACs in this study. Insoluble PACs have previously been described to occur in leaves and apexes from East African browse species and a variety of fruits.10−12 Insoluble PACs are often bound to plant components, such as fiber or protein, and, as a result, are not readily solubilized by aqueous extraction (i.e., juicing processes) or aqueous/organic solvent extraction. Therefore, insoluble PACs are incapable of being measured by the DMAC method. However, both soluble and insoluble PACs undergo an auto-oxidation reaction when heated (100 °C) in a butanol−HCl solution (95:5, v/v), liberating anthocyanidins that can be quantified by spectrophotometric methods. Reed reported on the application of the BuOH−HCl method for qualitative identification of insoluble PACs.10 However, the quantity of insoluble PACs was expressed on an absorbance (550 nm) equivalent per unit dry matter because an appropriate reference standard for converting absorbance (Abs550) to a weight equivalence was not available. PérezJiménez et al. used a commercially available carob pod ( Ceratonia siliqua) PAC standard containing high-molecularSpecial Issue: Advances in Polyphenol Chemistry: Implications for Nutrition, Health, and the Environment Received: Revised: Accepted: Published: A

June 13, 2019 July 3, 2019 July 5, 2019 July 5, 2019 DOI: 10.1021/acs.jafc.9b03696 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry weight PACs11−13 to quantify the amount of insoluble PACs in a variety of different fruits. The assumption of the author is that the carob pod PACs and the kinetics of the reaction in the BuOH−HCl assay were similar in all of the fruit samples that were evaluated. Considering the diversity of fruits and the heterogeneity of PACs (degree of polymerization, ratios of Ato B-type interflavan bonds, hydroxylation patterns, etc.) that is unique to each fruit, the use of carob pod PACs likely produces an inaccurate estimation of insoluble PACs in samples that are not of carob pod origin. To improve the accuracy of quantifying insoluble PACs across a diverse set of samples, customized PAC reference standards isolated from the fruit of interest and reflective of the natural structural heterogeneity of the PACs found in the fruit can be developed. We report, for the first time, the detection and quantification of insoluble PACs in cranberry solids. We also introduce, for the first time, an appropriate reference standard (c-PAC) that can be used in the BuOH−HCl assay to convert absorbance values to a weight basis.

Chromaflex) packed with Sephadex LH-20 that was previously swollen and washed in water and equilibrated with ethanol for 45 min at a flow rate of 4 mL/min.7 The column was sequentially eluted with 600 mL of ethanol, 600 mL of ethanol/methanol (1:1, v/v), and 600 mL of 80% aqueous acetone (v/v). The 80% aqueous acetone fraction was evaporated to dryness, solubilized in methanol, and defined as “cPAC” standard reference material. The concentration of PAC in the cPAC standard reference material was 90.13 mg of dry matter/mL, as determined by the DMAC method. 2.4. Characterization of the c-PAC Standard. Previously developed matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI−TOF MS) methods were applied to characterize the c-PAC standard reference material.7,14,15 An aliquot of the extract was evaporated to dryness and suspended in ethanol to a final concentration of approximately 20 mg/mL. An aliquot (0.5 μL) of this sample was mixed with 1 μL of 2,5-dihydroxybenzoic acid (50.0 mg/mL in ethanol) and placed in three different wells on the stainless-steel MALDI target. Mass spectra were collected on a Bruker Ultraflex III MALDI−TOF/TOF (Billerica, MA, U.S.A.), equipped with a Smart Beam, a two-stage gridless reflectron, and a LIFT cell. Mass spectra acquired in positive reflectron mode are the sum of spectra from different locations in each well with the deflection set at 550. Compass version 1.3 software controlled the instrument. An internal laboratory standard cranberry PAC preparation was used for calibration as previously described.7 FlexControl and FlexAnalysis (version 3.0, Bruker Daltonik GmbH, Bremen, Germany) were used for data acquisition and data processing, respectively. 2.5. Extraction of Soluble PACs and Drying of Insoluble Material. A total of nine commercial cranberry products were analyzed as previously described.6,7 Triplicate aliquots of each of the nine products were weighed (0.5 g) in 50 mL conical tubes. The tare weight of the centrifuge tube and cap were recorded prior to weighing the sample. A total of 5 mL of 2% acetic acid in deionized water (v/v) was added to each sample. Samples were vortexed and placed in an ultrasonic bath at room temperature for 10 min. After ultrasonic extraction, 100% acetone (15 mL) was added to each sample. Samples were vortexed, placed in an ultrasonic bath for 30 min, and subsequently centrifuged at 1840g and 20 °C for 10 min. The supernatant was collected and used to quantify soluble PACs by the DMAC assay. The centrifuge cap and tube containing the pellet remaining after centrifugation were placed in a forced air oven (40 °C), with the cap removed, and dried overnight. Alfaro-Viquez et al. have demonstrated that thermodegradation of PAC does not occur at 40 °C.16 The tube, cap, and pellet were weighed again. The difference between the original weight of the sample and the residual pellet reflect the quantity of sample that was solubilized during the extraction process. This difference in weight was used as a correction factor to convert insoluble PAC values determined on the residual pellet (milligrams of c-PAC equivalents per gram of residual pellet) to insoluble PAC values reported on an original weight (as received) basis (milligrams of c-PAC equivalent per gram of original sample). The dried residue was ground with a mortar and pestle in preparation for insoluble PAC analysis by the BuOH−HCl assay. 2.6. Preparation of c-PAC Calibration Standards for DMAC and BuOH−HCl Analyses. Cranberry PAC (c-PAC) standard working solutions with concentrations of 0, 25, 100, 250, 500, and 750 μg/mL were prepared in methanol for the BuOH−HCl analysis. Cranberry PAC (c-PAC) standard working solutions with concentrations of 16, 33, 49, 65, 81, and 98 μg/mL were prepared in 75% aqueous ethanol (v/v) for the DMAC analysis. 2.7. Quantification of Soluble PACs by the Method of DMAC. Quantification of soluble PACs was performed by the DMAC assay as previously reported.7,8 A total of 70 μL of blanks (75% aqueous ethanol, v/v), c-PAC standards, and samples was dispensed, in technical triplicates from each of the three extracted replicates, into individual wells on a polystyrene 96-well plate. The 0.1% DMAC solution was prepared by adding DMAC (0.025 g) to 25 mL of reaction buffer produced as follows: (37%) hydrochloric acid (125 mL) was added to 125 mL of distilled water and 750 mL of ethanol in a glass bottle and mixed. This 0.1% DMAC solution reagent must be

2. METHODS 2.1. Chemicals and Reagents. High-performance liquid chromatography (HPLC)-grade water was from a Millipore Milli-Q water purification system (Millipore, Bedford, MA, U.S.A.). HPLCgrade methanol (99.9%), Acetic acid (glacial), n-butanol, and acetone were from Fisher Scientific, Houston, TX, U.S.A. Ethanol (100%) was from Decon Labs (King of Prussia, PA, U.S.A.). DMAC and hydrochloric acid (37%) were from Sigma-Aldrich (St. Louis, MO, U.S.A.). Sephadex LH-20 was purchased from GE Healthcare (Uppsala, Sweden), and the cranberry (c-PAC) standard was produced by the method described below in section 2.3 and characterized as described below in section 2.4. As previously reported, the purity of c-PAC standard was estimated to be 99.0 ± 1.3% by the formaldehyde−HCl precipitation test, and elemental analysis showed 58.2% carbon, 4.7% hydrogen, and 37.3% oxygen.7 2.2. Sample Description. Nine commercially available whole cranberry (Vaccinium macrocarpon Ait.) fruit ingredients (dried cranberry fruits and dried cranberry powder) were procured and analyzed as received. The cranberry samples represent products that are currently being sold as foods and/or ingredients in the dietary supplement market. All products are derived from whole cranberry fruit without selective extraction processes to enrich soluble PAC composition. The specifics of the processes for the production of these products are proprietary information of the manufacturers. Cranberry (V. macrocarpon Ait.) fruit (cv. Stevens) was used to generate the c-PAC standard. The cranberry fruit was ground to a fine powder with liquid nitrogen and frozen at −80 °C until further use in preparation of the c-PAC standard. 2.3. Preparation of the c-PAC Standard. The c-PAC extraction procedure was adapted from previous research7 using a total weight of 1314 g of cranberry (V. macrocarpon Ait.) powder (151.15 g of dry matter). The powder was generated by pouring liquid nitrogen over the frozen cranberry fruit (cv. Stephens) and homogenizing to a powder in a 450 W commercial blender. For each 100 g of cranberry powder, 400 mL of 70% aqueous acetone (v/v) was added and extracted in an ultrasonic bath for 15 min. The extract was centrifuged at 400g and 15 °C for 10 min, and the supernatant was collected. The extraction was repeated 2 additional times, and the supernatants were combined. The extract was passed through filter paper (Whatman no. 1). Acetone was removed by rotoevaporation under vacuum at 35 °C. The remaining suspension was solubilized in 100 mL of ethanol. The extract was then centrifuged at 13416g and 0 °C for 10 min to eliminate insoluble material. The ethanolic supernatant was collected and reduced to 50 mL to be used to isolate c-PAC by chromatography on Sephadex LH-20. The 50 mL ethanolic cranberry extract was loaded on glass columns (2.5 cm inner diameter × 60 cm length, Kontes, B

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Journal of Agricultural and Food Chemistry Table 1. Conversion of a Sample Absorbance Value to Milligrams of c-PAC Equivalents per Gram of Sample B (μg/mL)

A (abs)

D

V (mL)

R (mg)

W (mg)

M (mg)

Y (550 nm)

insoluble PACs (mg/g)

sample average (mg/g)

27.005

0.0239

2

5.00

415.91

496.90

499.32

422.68

506.92

0.973 0.894 0.794 0.800 0.873 0.848 0.820 0.817 0.845

57.8 53.5 50.3 51.0 52.3 52.0 51.5 50.5 51.4

52.3

417.11

5.35 5.32 5.04 5.00 5.30 5.19 5.06 5.14 5.22

Figure 1. PAC oligomers ranging from 2 to 11 degrees of polymerization with structural variation in the nature of the interflavan bond detected by positive reflectron mode MALDI−TOF MS. made fresh daily. A multichannel pipet was used to dispense 210 μL of 0.1% DMAC solution into each well, including blanks. Immediately after the addition of the DMAC solution, the plate was placed into a plate reader (Spectromax) and shaken for 10 s at 600 shakes per minute. Absorbance readings at 640 nm were taken every 60 s for 0.5 h. The corresponding blank absorbance readings at each time point were subtracted from the standard and sample wells. Results were collected at the time of maximum absorbance and expressed as milligrams of c-PAC equivalents per gram of original sample, as previously described.7,8 2.8. Quantification of Insoluble PACs by the Method of BuOH−HCl. Quantification of insoluble PAC in the sample residue after extraction was determined by modifications of the methods described by Bate-Smith17 and Reed.10 A total of 500 μL of each of the five c-PAC reference standard dilutions (described in section 2.6) and a blank (methanol) were pipetted in individual test tubes (12.7 mm outer diameter × 100 mm). The final concentrations of the reference standards in the total reaction volume (5.5 mL) are 2.29, 9.18, 22.61, 45.56, and 68.17 μg/mL. In triplicate, 5 mg of each of the three replicates of sample residues (remaining after extraction) were weighed into individual test tubes. A total of 5 mL of 5% concentrated HCl in n-butanol (v/v) solution was added to all tubes. The tubes were vortexed and placed in a preheated (100 °C) hot block (13 mm diameter wells). A glass marble was placed on top of each tube to prevent evaporation of the reaction solvent. Samples were heated (100 °C) for 1 h. Tubes were removed from the hot block and placed in an ice bath until the blank reached 22 °C. Absorbance of the

supernatant was recorded at 550 nm. The recorded absorbance values of the c-PAC standard were used to generate a standard curve based on the relationship between the absorbance and concentration. The slope of the standard curve was used to convert the absorbance values of the samples to a weight basis. The concentration of insoluble PAC (mg/g) in the samples was calculated using the following equation: y−A iR y =jjj × × DV zzz ÷ M × 1000 μg/mg B kW {

where W is the initial sample mass (mg), R is the sample mass following soluble extraction (mg), y is the absorbance at 550 nm (abs), A is the y intercept (abs), B is the slope (μg/mL), D is the dilution factor, V is the total volume (mL), and M is the residue sample mass (mg). The calculation accounts for the weight of sample that was lost (solubilized) during the extraction process. The final results are reported as milligrams of c-PAC equivalence per gram of original sample weight. An example of the application of the equation is shown in Table 1.

3. RESULTS AND DISCUSSION 3.1. MALDI−TOF MS Characterization of the c-PAC Standard. Positive reflectron mode MALDI−TOF MS detected a series of PAC oligomers that ranged from 2 to 11 degrees of polymerization (Figure 1) with structural variation in the nature of the interflavan bond (Figure 2). The predominant mass at each degree of polymerization (Δ288 C

DOI: 10.1021/acs.jafc.9b03696 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Table 2. Quantification of Soluble and Insoluble PACs in Cranberry Products by the Methods of DMAC and BuOH− HCl, Separatelya sample sample description 1 2 3 4 5 6 7 8 9

cranberry powder cranberry powder cranberry powder cranberry powder cranberry powder cranberry powder dried cranberry dried cranberry dried cranberry

DMAC soluble PACs (mg/g)

BuOH−HCl insoluble PACs (mg/g)

30.40 ± 1.09

16.06 ± 1.13

32.45 ± 0.91

14.65 ± 1.82

29.91 ± 0.95

34.43 ± 1.02

29.87 ± 0.56

47.34 ± 1.99

34.75 ± 0.76

40.60 ± 1.56

28.48 ± 0.33

52.25 ± 2.28

33.50 ± 0.58

16.63 ± 0.78

26.15 ± 0.52

20.03 ± 1.06

44.78 ± 2.07

18.39 ± 0.36

a

Results are expressed as milligrams of c-PAC equivalents per gram of original sample.

Figure 2. Representative structure of a cranberry PAC trimer [epicatechin−(4β-8)-epicatechin−(4β-8,2β-O-7)-epicatechin]. Variation in degree of polymerization, position, and number of “A-type” versus “B-type” interflavan bonds leads to large structural heterogeneity among cranberry PACs.18

throughout the ripening process. Bordiga et al. measured the compositional changes that occur in both soluble and insoluble PACs in the peel and pulp during fruit maturation, showing a decrease in the PAC concentration as the fruit ripened.19 The development of the BuOH−HCl method using the c-PAC reference standard provides the analytic tools for growers and processors of cranberry products to track both the soluble and insoluble PAC components and understand how these classes of compounds are partitioned on the basis of their manufacturing processes. In the estimation of insoluble PACs, it is assumed that the reaction kinetics of the c-PAC standard are similar to the reaction kinetics of the insoluble PACs associated with extracted cranberry residue. As a result of the association of PACs with insoluble material (i.e., insoluble fiber), it is likely that there is an underestimation of insoluble PACs quantified in BuOH−HCl even with the use of the c-PAC standard because not all of the anthocyanidin leaving groups are liberated. Additional research to test the assumption of the reaction kinetics of insoluble PACs and the accuracy of the BuOH−HCl method is required and will likely lead to improvements in the accurate quantification of insoluble PACs. As previously mentioned, PACs in cranberry fruits and commercial products are partitioned between soluble (extractable) and/or insoluble (non-extractable). The history of how the cranberry sample was handled and processed dictates the relative proportioning of these two classes of PACs. Products such as cranberry juice and spray dried juice powders contain almost exclusively soluble PACs, while both soluble and insoluble PACs can be found in fresh and frozen cranberry fruit, whole cranberry fruit products, such as sweetened dried cranberries, and juice process co-products, such as press cake, seeds, and skins. There are numerous nutritional supplement ingredients and products on the market that are formulated to include both soluble and insoluble PACs. Insoluble PACs cannot be quantified by the DMAC assay as a result of their non-extractable nature and, thus, represent an underappreciated and uncharacterized proanthocyanidin component of many cranberry products.

amu) is representative of a PAC structure that contains one or more A-type interflavan bond. These observed masses and mass distribution are consistent with previously published PAC composition of cranberry fruit and the c-PAC standard.7,8 Our previous chromatographic analysis of the c-PAC standard also indicates that no other classes of polyphenols (anthocyanins, flavonols, and hydroxycinnamic acids) were present.7 These results indicate that the c-PAC standard is reflective of the structural heterogeneity of PACs found in cranberry fruit and is, thus, an appropriate standard reference material for use in quantifying both soluble PACs (DMAC analysis) and insoluble PACs (BuOH−HCl analysis). 3.2. Quantification of Soluble and Insoluble PACs. The mean and standard deviation (n = 9; 3 extracted residues × 3 technical replicates per extract) of soluble PACs for the nine commercial products tested ranged from 28.48 ± 0.33 to 44.78 ± 2.07 mg of c-PAC/g (Table 2). The mean and standard deviation (n = 9; 3 extracted residues × 3 technical replicates per residue) of insoluble PACs for the nine commercial products tested ranged from 14.65 ± 1.82 to 52.25 ± 2.28 mg of c-PAC/g (Table 2). The relatively low standard deviation demonstrates that both the DMAC and BuOH−HCl methods are precise and repeatable. The larger average standard deviation in the BuOH−HCl results may be attributed to subsampling bias when weighing small quantities (5 mg) of the non-homogenous fibrous residues from cranberry products. It is important to note that the total amount of soluble PACs, the total amount of insoluble PACs, and the relative proportion of soluble to insoluble PACs is not consistent across all samples. The variation in partitioning of the soluble and insoluble PACs may be attributed to variation in cranberry fruit (variety, harvest date, and location), storage conditions, and processing conditions. This phenomenon has been demonstrated by the characterization of persimmon fruit D

DOI: 10.1021/acs.jafc.9b03696 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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ACKNOWLEDGMENTS The authors thank Fruit d’Or for providing commercial cranberry products for the study.

There is an ever-increasing diversity of cranberry products entering the food and dietary supplement markets. As a result of this trend, raw ingredient providers, formulators, marketers, and health researchers are increasingly dependent upon methods for authentication, standardization, and efficacy evaluation of products. The cranberry industry has adopted the DMAC analysis for the quantification of soluble PACs, HPLC for the identification and quantification of flavonols, anthocyanins, and hydroxycinnamic acids, and MALDI−TOF MS for the authentication of specific PAC structural features, such as A-type interflavan bonds. Furthermore, the cranberry marketplace has recognized the utility of the c-PAC reference standard to provide more accurate quantification of PACs than the procyanidin A2 dimer.7,8 Through the use of an appropriate reference standard, the BuOH−HCl assay can be used to quantify insoluble PACs. The ability to quantify soluble and insoluble PACs will enable manufactures to better control the effects that harvesting, storage, and processing have on PAC composition. Furthermore, the quantification of insoluble PACs associated with the fiber fractions of cranberry products will enable researchers to develop and test new hypotheses on the bioactivity of this class of PAC in relation to health and nutrition. The current literature focuses on the health-related outcomes of soluble PACs, whereas the health impact of insoluble PACs remains underrepresented. Insoluble PACs primarily affect the colon as a result of their ability to remain intact throughout the gastrointestinal tract while associated with fiber. Once in the colon, the fiber ferments and the liberated PACs are speculated to increase colonic antioxidant capacity, increase production of absorbable metabolites, and decrease tumorigenesis in the intestine.20 The ability to quantify insoluble PACs with a proper reference standard will further this research and allow for greater understanding of these outcomes. The results of this current work indicate that the BuOH− HCl assay, when used with an appropriate standard (c-PAC), can be used to quantify insoluble PACs. The adoption of the BuOH−HCl method for insoluble PACs will provide a more comprehensive characterization of cranberry products and will impact numerous aspects of the cranberry and cranberryrelated industries.

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REFERENCES

(1) Foo, L.; Lu, Y.; Howell, A.; Vorsa, N. A-Type Proanthocyanidin trimers from cranberry that inhibit adherence of Uropathogenic PFimbriated Escherichia coli. J. Nat. Prod. 2000, 63, 1225−1228. (2) Howell, A.; Reed, J.; Krueger, C.; Winterbottom, R.; Cunningham, D.; Leahy, M. A-type cranberry proanthocyanidins and uropathogenic bacterial anti-adhesion activity. Phytochemistry 2005, 66, 2281−2291. (3) Neto, C.; et al. MALDI-TOF MS characterization of proanthocyanidins from cranberry fruit Vaccinium macrocarpon that inhibit tumor cell growth and matrix metalloproteinase expression in vitro. J. Sci. Food Agric. 2006, 86, 18−25. (4) Reed, J.; Krueger, C.; Vestling, M. MALDI-TOF mass spectrometry of oligomeric food polyphenols. Phytochemistry 2005, 66, 2248−2263. (5) Krueger, C.; Reed, J.; Feliciano, R.; Howell, A. Quantifying and characterizing proanthocyanidins in cranberries in relation to urinary tract health. Anal. Bioanal. Chem. 2013, 405, 4385−4395. (6) Prior, R.; Fan, E.; Ji, H.; Howell, A.; Nio, C.; Payne, M.; Reed, J. Multi-laboratory validation of a standard method for quantifying proanthocyanidins in cranberry powders. J. Sci. Food Agric. 2010, 90, 1473−1478. (7) Feliciano, R.; Shea, M.; Shanmuganayagam, D.; Krueger, C.; Howell, A.; Reed, J. Comparison of isolated cranberry (Vaccinium macrocarpon Ait.) Proanthocyanidins to Catechin and Procyanidins A2 and B2 for Use as Standards in the 4- (Dimethylamino)cinnamaldehyde Assay. J. Agric. Food Chem. 2012, 60, 4578−4585. (8) Krueger, C.; Chesmore, N.; Chen, X.; Parker, J.; Khoo, C.; Marais, J.; Shanmuganayagam, D.; Crump, P.; Reed, J. Critical reevaluation of the 4-(dimethylamino)cinnamaldehyde assay: Cranberry proanthocyanidin standard is superior to procyanidin A2 dimer for accurate quantification of proanthocyanidins in cranberry products. J. Funct. Foods 2016, 22, 13−19. (9) Wallace, T.; Giusti, M. Evaluation of parameters that affect the 4dimethylaminocinnamaldehyde assay for flavanols and proanthocyanidins. J. Food Sci. 2010, 75, C619−C625. (10) Reed, J. Relationships among soluble phenolics, insoluble proanthocyanidins and fiber in east African browse species. J. Range Manage. 1986, 39, 5. (11) Pérez-Jiménez, J.; Arranz, S.; Saura-Calixto, F. Proanthocyanidin content in foods is largely underestimated in the literature data: An approach to quantification of the missing proanthocyanidins. Food Res. Int. 2009, 42, 1381−1388. (12) Pérez-Ramírez, I.; Reynoso-Camacho, R.; Saura-Calixto, F.; Pérez-Jiménez, J. Comprehensive characterization of extractable and nonextractable phenolic compounds by high-performance liquid chromatography−electrospray ionization−quadrupole time-of-flight of a grape/pomegranate pomace dietary supplement. J. Agric. Food Chem. 2018, 66, 661−673. (13) Perez-Jimenez, J.; Saura-Calixto, F. Macromolecular antioxidants or non-extractable polyphenols in fruit and vegetables: Intake in four European countries. Food Res. Int. 2015, 74, 315−323. (14) Krueger, C.; Dopke, N.; Treichel, P.; Folts, J.; Reed, J. Matrixassisted laser desorption/ionization time-of-flight mass spectrometry of Polygalloyl Polyflavan-3-ols in grape seed extract. J. Agric. Food Chem. 2000, 48, 1663−1667. (15) Feliciano, R.; Krueger, C.; Shanmuganayagam, D.; Vestling, M.; Reed, J. Deconvolution of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry isotope patterns to determine ratios of A-type to B-type interflavan bonds in cranberry proanthocyanidins. Food Chem. 2012, 135, 1485−1493. (16) Alfaro-Viquez, E.; Esquivel-Alvarado, D.; Madrigal-Carballo, S.; Krueger, C.; Reed, J. Cranberry proanthocyanidins-chitosan hybrid nanoparticles as a potential inhibitor of extra-intestinal pathogenic

AUTHOR INFORMATION

Corresponding Author

*Telephone: +1-608-423-1327. E-mail: ckrueger@phyto-sol. com. ORCID

Christian G. Krueger: 0000-0003-4242-9596 Funding

All mass spectrometry work was performed on a Bruker ULTRAFLEX III, which was partially funded by the National Institutes of Health (NIH) National Center for Research Resources (NCRR) 1S10RR024601-01 grant to the Department of Chemistry, University of WisconsinMadison. Notes

The authors declare the following competing financial interest(s): Christian G. Krueger and Jess D. Reed have ownership interest in Complete Phytochemical Solutions, LLC, and in full disclosure, their affiliation with this company is acknowledged in the author affiliation. E

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Journal of Agricultural and Food Chemistry Escherichia coli invasion of gut epithelial cells. Int. J. Biol. Macromol. 2018, 111, 415−420. (17) Bate-Smith, E. Phytochemistry of proanthocyanidins. Phytochemistry 1975, 14, 1107−1113. (18) Foo, L.; Lu, Y.; Howell, A.; Vorsa, N. A-Type proanthocyanidin trimers from cranberry that inhibit adherence of uropathogenic Pfimbriated Escherichia coli. J. Nat. Prod. 2000, 63, 1225−1228. (19) Bordiga, M.; Travaglia, F.; Giuffrida, D.; Mangraviti, D.; Rigano, F.; Mondello, L.; Arlorio, M.; Coïsson, J. Characterization of peel and pulp proanthocyanidins and carotenoids during ripening in persimmon “Kaki Tipo” cv, cultivated in Italy. Food Res. Int. 2019, 120, 800−809. (20) Pérez-Jiménez, J.; Díaz-Rubio, M.; Saura-Calixto, F. Nonextractable polyphenols, a major dietary antioxidant: Occurrence, metabolic fate and health effects. Nutr. Res. Rev. 2013, 26, 118−129.

F

DOI: 10.1021/acs.jafc.9b03696 J. Agric. Food Chem. XXXX, XXX, XXX−XXX