Article pubs.acs.org/JAFC
Separation and Characterization of Phenolic Compounds from U.S. Pecans by Liquid Chromatography−Tandem Mass Spectrometry Katherine S. Robbins,† Yuanyuan Ma,† M. Lenny Wells,‡ Phillip Greenspan,§ and Ronald B. Pegg*,† †
Department of Food Science & Technology, College of Agricultural and Environmental Sciences, The University of Georgia, 100 Cedar Street, Athens, Georgia 30602-2610, United States ‡ Department of Horticulture, College of Agricultural and Environmental Sciences, The University of Georgia, 4604 Research Way, Tifton, Georgia 31793-5766, United States § Department of Pharmaceutical & Biomedical Sciences, College of Pharmacy, The University of Georgia, 250 W. Green Street, Athens, Georgia 30602, United States S Supporting Information *
ABSTRACT: The phenolic acids and proanthocyanidins (PACs) of pecans possess bioactive properties, which might be useful in retarding the onset of and ameliorating the status of certain chronic disease states. There is a general lack of information in the literature regarding such compounds, especially the PACs. Crude phenolic extracts pooled from eight commercially significant cultivars were selected based on their relatively high antioxidant capacities. The pooled extracts were separated via Sephadex LH20 column chromatography into five ethanolic low-molecular-weight (LMW) fractions and one acetonic high-molecular-weight (HMW) fraction. The preparations were then characterized using RP-HPLC-ESI-MS/MS and diol-phase HPLC-ESI-MS/MS in order to determine the key constituents present in the LMW and HMW fractions, respectively. As previously observed in pecan nutmeat, ellagic acid and (+)-catechin were found to be the major phenolics in the LMW fractions. The last eluting LMW fraction did not contain phenolic acids; rather it possessed PAC monomers and dimers. The HMW fraction comprised a majority of its PACs as dimers; yet, monomers, trimers, tetramers, pentamers, and hexamers were also separated and characterized. KEYWORDS: pecans, proanthocyanidins (PACs), Sephadex LH-20, RP-HPLC, diol-phase-HPLC, ESI-MS/MS
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INTRODUCTION Pecans [Carya illinoinensis (Wangenh.) K. Koch] and other tree nuts are receiving increased attention due to their purported health benefits1−4 and a qualified health claim approved more than 10 years ago by the U.S. Food and Drug Administration. Most reports associate these benefits with healthful lipid profiles (i.e., MUFAs, PUFAs, α-tocopherol, and γ-tocopherol), but more recently attention has turned toward the favorable phenolic profiles that tree nuts possess. Endogenous pecan phenolics can be segregated into two main classes, namely, phenolic acids (e.g., ellagic and gallic acids as well as their derivatives) and condensed tannins/proanthocyanidins (PACs) with varying degrees of polymerization (DP).5 Characterization of these important classes, especially the PACs, is lacking in the literature.6 PACs, or condensed tannins, are a type of phenolic polymer. There are two main types, that is, procyanidins and prodelphinidins. Procyanidins, the most prevalent PAC found in foods, are composed of (−)-epicatechin monomers, whereas the prodelphinidins comprise (−)-epigallocatechin monomers.7 PACs are usually not classified based on the individual compound type; rather, they are centered on the DP that exist.8 The DP can range anywhere from 2 (i.e., dimers) to 10+ (i.e., polymers). The antioxidant properties and antimutagenic activity of PACs can vary based upon structure and DP.7 Pecan PACs and their flavan-3-ol monomers have shown potential for important health-related benefits in vivo.6 In a randomized controlled trial employing a crossover design with © 2014 American Chemical Society
a one-week washout period between treatments, Hudthagosol et al.6 had 16 healthy men and women (23−44 years, BMI 22.7 ± 3.4) consume a test meal composed of either whole pecans, blended pecans, or an isocaloric meal of equivalent macronutrient composition. Blood was sampled at baseline and at intervals up to 24 h postingestion. Plasma antioxidant capacities and the levels of γ-tocopherol and epigallocatechin gallate (EGCG) from the panelists’ blood were determined; each increased significantly (p < 0.05) postprandially. While the flavan-3-ol monomers can be absorbed in the small intestine, there is evidence that all PACs with a DP of 3 and higher may not be able to be properly absorbed.9 If this is indeed the case, larger PACs can still participate in contributing a health benefit to humans, as these compounds can be broken down into smaller secondary metabolites by colonic microflora10 and then be absorbed. In this study, we employed Sephadex LH-20 column chromatography to fractionate pecan phenolic acids, their derivatives, and PACs. Once separated, these fractions were characterized by the total phenolics content (TPC) assay, UV spectral analysis, and RP-HPLC-ESI-MS/MS characterization of the low-molecular-weight (LMW) fractions and diol-phase Received: Revised: Accepted: Published: 4332
February April 11, April 15, April 16,
20, 2014 2014 2014 2014
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analyzed. A total lipid and phenolic extraction was completed in triplicate for each of the 18 pecan cultivars. Sample Selection and Extract Separation. On the basis of preliminary data (results not shown here), the eight cultivars with the best performance based on the H-ORACFL assay were selected for use in this study. A 240 mg portion of each crude pecan extract was taken and pooled. The resultant extract (1.92 g) was mixed in a small volume of ∼75% (v/v) ethanol and sonicated to ensure its complete dissolution. The sample was then applied via a Pasteur pipet to the top of a chromatographic column packed with Sephadex LH-20 (bead size: 25−100 μm; Chromaflex column, 30 × 400 mm [i.d. × length], Kontes, Vineland, NJ, USA), and flow was conducted by gravity. The LMW phenolic compounds (molecular weight 10mers constituted 3.4, 8.5, 5.3, 20.5, 17.0, and 45.2%, respectively, in pecans. It should be noted that in the present study we did not find any PACs larger than hexamers. With the removal of the 7−10mers and >10 mers from the Gu et al.8 data set, the monomers, dimers, trimers, and 4−6mers now account for 9.2, 22.5, 13.9, 54.3%, respectively. In comparison to the prevalence of tetramers through hexamers in the former study, the present one found dimers to be most abundant. Earlier methods of PAC analysis (i.e., TLC analysis, RP-HPLC, and early normalphase HPLC methods) were discredited for failing to separate compounds with DP > 4.27 In preparing for this research, standards were run with DP of 8, 9, and even 10 with excellent resolution and detection on the diol-phase column and the fluorescence detector. For this reason, we believe that any PACs with DP > 7 would have been visible with this methodology and no suppression of ions occurred. In a study by Kelm et al.,15 in which the diol-phase HPLC methodology was first introduced, there was also excellent resolution up to a DP = 14. On the basis of Kelm et al.15 and the employment of the diol-phase column, we believe that PACs with higher DP would be properly resolved if they had been present in the defatted pecan acetonic extract. This was further confirmed by the MS data. No ions corresponding to PACs with a DP higher than 6 were identified in the sample. While the authors believe that all of the PACs containing (+)-catechin and (−)-epicatechin monomers were identified and quantified, there may be some PACs that are not properly captured with the fluorescence detector. Lazarus et al.28 points out that fluorescence detection cannot be used for the identification of PACs with gallocatechin and gallic ester components. Further HPLC investigation of these compounds using diol-phase diode-array detection is necessary. Additional information regarding specific composition, monomer position, and length of the PACs could be acquired using the thiolysis preparation technique coupled with HPLC characterization. In thiolysis, individual PAC units are separated into end units and interior units through an acid-catalyzed cleavage of the interflavan bonds.29 After cleavage, the interior unit intermediates are reacted with toluene α-thiol, forming thiol-flavonol adducts, and the end units remain as flavanol monomers.29,30 These thiol adducts can then be separated and identified with HPLC analysis to give a better picture of the average DP of the PACs, as well as the position and type of the monomeric units.30 We are not aware of any literature studies that have investigated pecan PACs using a thiolysis step. It has also been noted that when PACs are separated on Sephadex LH-20 via isocratic elution, the first compounds to elute will be those PACs with smaller MWs and the heavier ones will elute last.31 A possible reason for a reduction in the quantity of HMW PACs in our study could be that a sufficient quantity of the aqueous acetone was not employed to elute all PACs from the column. However, based on the mass balance performed in this work, it is not possible that this could account for the extreme differences observed. A total of 1.92 g of crude extract was loaded onto the Sephadex LH-20 column, and a mass of 1.90 g comprising the various fractions was recovered after lyophilization. This signifies that 99% of the crude extract applied to the column was recovered via the chromatography and that very small quantities of higher DP, if they existed, might remain on the Sephadex LH-20 column. Unfortunately the Gu et al.8 study is the only literature source that quantifies and characterizes PAC components of pecan nutmeat. Further
Table 4. Characterization and Quantification of the Tannin Fraction from a Pecan Crude Acetonic Extract Isolated by Sephadex LH-20 Column Chromatography Using 50% (v/v) Aqueous Acetonea degrees of polymerization (DP)
pooled cultivarsb
1 2 3 4−6 total
0.3 47.3 21.1 14.8 83.5
a
Characterized by diol-phase HPLC and quantification completed using commercial standards with varying degrees of polymerization. b All quantities are expressed as mg compound/g tannin fraction.
fraction(s). These findings demonstrate that the tannin fractions, in particular those with higher DP, may play a role in determining the observed in vitro antioxidant activity of a sample. A deeper examination of the PAC composition was completed using diol-phase HPLC-ESI-MS/MS analyses. After completing a full scan over the mass range of 200 to 3000 Da, a search of common PAC mass ions from the literature20,26 was undertaken: nine compounds were identified in the acetonic fraction (i.e., fraction VI; see Table 5). All of the Table 5. PAC Compounds Present in the Tannin Fraction Isolated from Eight Pooled Pecan Cultivars tRa
DPb
unit typesc
linkage
[M − H]−
6.5 9.7 11.0 13.1 14.2 16.0 16.3 18.0 19.0
2 3 3 4 4 5 5 6 6
2 (epi)catechin 3 (epi)catechin 2 (epi)catechin and 1 (epi)gallocatechin 4 (epi)catechin 3 (epi)catechin and 1 (epi)gallocatechin 5 (epi)catechin 4 (epi)catechin and 1 (epi)gallocatechin 5 (epi)catechin and 1 (epi)gallocatechin 4 (epi)catechin and 2 (epi)gallocatechin
B B B B B B B B B
577 865 881 1153 1169 1441 1457 1745 1761
a
Retention time determined from diol-phase HPLC-ESI-MS analyses. Degrees of polymerization. cCross-checking with compounds identified in nut skins, as reported by Sarnoski et al.20 and Monagas et al.26 b
compounds present in the sample exhibited a B-type linkage, which was to be expected based on literature reports of pecan PACs.8 Interestingly, the tannin fraction possessed both procyanidin and prodelphinidin compounds. Two PACs, one procyanidin and one prodelphinidin, were detected for each DP with the exception of the dimer, which had a single identification, and the hexamers, which were both prodelphinidins. The single dimer is composed of two (epi)catechin units based on a tR of 6.5 min and a molecular ion [M − H]− at m/z 577. The two trimers identified in the pecan sample had molecular ions [M − H]− at m/z 865 and 881 and eluted at a tR of 9.7 and 11.0 min, respectively. On the basis of the [M − H]−, these compounds were identified as possessing 3 (epi)catechin units and 2 (epi)catechin + 1 (epi)gallocatechin unit. The two detected trimers differed in their molecular ions by 16 amu, and this trend continued for the three larger DP of m/z at 1153−1169, 1441−1457, and 1745−1761. Gu et al.8 stated that PACs are present in numerous different food types including pecans. These authors reported that the 4339
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(2) Kris-Etherton, P. M.; Zhao, G.; Binkoski, A. E.; Coval, S. M.; Etherton, T. D. The effects of nuts on coronary heart disease risk. Nutr. Rev. 2001, 59, 103−111. (3) Blomhoff, R.; Carlsen, M. H.; Andersen, L. F.; Jacobs, D. R., Jr. Health benefits of nuts: potential role of antioxidants. Br. J. Nutr. 2006, 96, S52−S60. (4) Sabaté, J.; Ros, E.; Salas-Salvadó, J. Nuts: nutrition and health outcomes. Br. J. Nutr. 2006, 96, S1−S2. (5) Villarreal-Lozoya, J. E.; Lombardini, L.; Cisneros-Zevallos, L. Phytochemical constituents and antioxidant capacity of different pecan [Carya illinoinensis (Wangenh.) K. Koch] cultivars. Food Chem. 2007, 102, 1241−1249. (6) Hudthagosol, C.; Haddad, E. H.; McCarthy, K.; Wang, P.; Oda, K.; Sabaté, J. Pecans acutely increase plasma postprandial antioxidant capacity and catechins and decrease LDL oxidation in humans. J. Nutr. 2011, 141, 56−62. (7) Rigaud, J.; Escribano-Bailon, M. T.; Prieur, C.; Souquet, J.-M.; Cheynier, V. Normal-phase high-performance liquid chromatographic separation of procyanidins from cacao beans and grape seeds. J. Chromatogr. A 1993, 654, 255−260. (8) Gu, L.; Kelm, M. A.; Hammerstone, J. F.; Beecher, G.; Holden, J.; Haytowitz, D.; Gebhardt, S.; Prior, R. L. Concentrations of proanthocyanidins in common foods and estimations of normal consumption. J. Nutr. 2004, 134, 613−617. (9) Holt, R. R.; Lazarus, S. A.; Sullards, M. C.; Zhu, Q. Y.; Schramm, D. D.; Hammerstone, J. F.; Fraga, C. G.; Schmitz, H. H.; Keen, C. L. Procyanidin dimer B2 [epicatechin-(4β-8)-epicatechin] in human plasma after the consumption of a flavanol-rich cocoa. Am. J. Clin. Nutr. 2002, 76, 798−804. (10) Déprez, S.; Brezillon, C.; Rabot, S.; Philippe, C.; Mila, I.; Lapierre, C.; Scalbert, A. Polymeric proanthocyanidins are catabolized by human colonic microflora into low-molecular-weight phenolic acids. J. Nutr. 2000, 130, 2733−2738. (11) Wu, X.; Beecher, G. R.; Holden, J. M.; Haytowitz, D. B.; Gebhardt, S. E.; Prior, R. L. Lipophilic and hydrophilic antioxidant capacities of common foods in the United States. J. Agric. Food Chem. 2004, 52, 4026−4037. (12) Craft, B. D.; Kosińska, A.; Amarowicz, R.; Pegg, R. B. Antioxidant properties of extracts obtained from raw, dry-roasted, and oil-roasted US peanuts of commercial importance. Plant Foods Hum. Nutr. 2010, 65, 311−318. (13) Swain, T.; Hillis, W. E. The phenolic constituents of Prunus domestica. I.−The quantitative analysis of phenolic constituents. J. Sci. Food. Agric. 1959, 10, 63−68. (14) Srivastava, A.; Greenspan, P.; Hartle, D. K.; Hargrove, J. L.; Amarowicz, R.; Pegg, R. B. Antioxidant and anti-inflammatory activities of polyphenolics from southeastern U.S. range blackberry cultivars. J. Agric. Food Chem. 2010, 58, 6102−6109. (15) Kelm, M. A.; Johnson, J. C.; Robbins, R. J.; Hammerstone, J. F.; Schmitz, H. H. High-performance liquid chromatography separation and purification of cacao (Theobroma cacao L.) procyanidins according to degree of polymerization using a diol stationary phase. J. Agric. Food Chem. 2006, 54, 1571−1576. (16) Boulekbache-Makhlouf, L.; Meudec, E.; Chibane, M.; Mazauric, J.-P.; Slimani, S.; Henry, M.; Cheynier, V.; Madani, K. Analysis by high-performance liquid chromatography diode array detection mass spectrometry of phenolic compounds in fruit of Eucalyptus globulus cultivated in Algeria. J. Agric. Food Chem. 2010, 58, 12615−12624. (17) de la Rosa, L. A.; Alvarez-Parrilla, E.; Shahidi, F. Phenolic compounds and antioxidant activity of kernels and shells of Mexican pecan (Carya illinoinensis). J. Agric. Food Chem. 2011, 59, 152−162. (18) Mertz, C.; Cheynier, V.; Günata, Z.; Brat, P. Analysis of phenolic compounds in two blackberry species (Rubus glaucus and Rubus adenotrichus) by high-performance liquid chromatography with diode array detection and electrospray ion trap mass spectrometry. J. Agric. Food Chem. 2007, 55, 8616−8624. (19) Fracassetti, D.; Costa, C.; Moulay, L.; Tomás-Barberán, F. A. Ellagic acid derivatives, ellagitannins, proanthocyanidins and other phenolics, vitamin C and antioxidant capacity of two powder products
and more extensive characterization of these compounds is necessary to determine more information about the contents of pecans, in particular the HMW PACs. By utilizing exact mass HPLC/MS technologies, one might be able to further elucidate the structure of these compounds in pecans from various growing locations and cultivar types. HPLC/MSn analysis has previously been used to gain excellent information regarding the PAC profile of peanut skins.20 Researchers aim to quantify and characterize phenolic compounds in natural products due to their beneficial health benefits. The PAC compounds have been shown to remain intact in the low pH environment of the stomach and arrive to the small intestine intact.32 In this form, the PACs are unable to be absorbed in large quantities.10 However, colon microflora is able to breakdown PAC polymers to smaller metabolites that can be absorbed and utilized.10 The full benefit of these metabolites is not fully understood, thereby making predictions of the biological activity of higher degree polymers difficult. Hudthagosol et al.6 demonstrated adroitly that concentrations of the main PAC monomers, (+)-catechin and (−)-epicatechin, increased in the plasma of individuals after pecan consumption; these results are encouraging. Further research on the bioaccessibility (i.e., the extent of release of nutrients/ phytochemicals from the food matrix during digestion) and the bioavailability (i.e., the extent of absorption and utilization of released nutrients/phytochemicals for normal physiological functions) of phenolic constituents and PACs of pecans need to be investigated to gain better insight into the role of these compounds in disease prevention.
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ASSOCIATED CONTENT
S Supporting Information *
This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: 706-542-1099. Fax: 706-542-1050. E-mail: pegg@uga. edu. Funding
The authors would like to acknowledge the Georgia Agricultural Commodity Commission for Pecans (GACCP) and the USDA-NIFA-SCRI Award No. 2011-51181-30674 for funding this research. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Use of the Proteomics and Mass Spectrometry (PAMS) core facility under the direction of Dr. Dennis Phillips of UGA’s Department of Chemistry is greatly appreciated; in particular, thanks are extended to Dr. Phillips for assisting Y.M. with the LC-MS/MS analyses. The authors would also like to acknowledge Dr. Agnieszka Kosińska-Cagnazzo of the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences for her assistance with setting up the diolphase HPLC analyses.
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REFERENCES
(1) Hu, F. B.; Stampfer, M. J. Nut consumption and risk of coronary heart disease: A review of epidemiologic evidence. Curr. Atheroscler. Rep. 1999, 1, 204−209. 4340
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from camu-camu fruit (Myrciaria dubia). Food Chem. 2013, 139, 578− 588. (20) Sarnoski, P. J.; Johnson, J. V.; Reed, K. A.; Tanko, J. M.; O’Keefe, S. F. Separation and characterisation of proanthocyanidins in Virginia type peanut skins by LC−MSn. Food Chem. 2012, 131, 927− 939. (21) Bolling, B. W.; Chen, C.-Y. O.; McKay, D. L.; Blumberg, J. B. Tree nut phytochemicals: composition, antioxidant capacity, bioactivity, impact factors. A systematic review of almonds, Brazils, cashews, hazelnuts, macadamias, pecans, pine nuts, pistachios and walnuts. Nutr. Res. Rev. 2011, 24, 244−275. (22) Malik, N. S. A.; Perez, J. L.; Lombardini, L.; Cornacchia, R.; Cisernos-Zevallos, L.; Bradford, J. Phenolic compounds and fatty acid composition of organic and conventional grown pecan kernels. J. Sci. Food Agric. 2009, 89, 2207−2213. (23) Senter, S. D.; Horvat, R. J.; Forbus, W. R. Relation between phenolic acid content and stability of pecans in accelerated storage. J. Food Sci. 1980, 45, 1380−1382, 1392. (24) John, J. A.; Shahidi, F. Phenolic compounds and antioxidant activity of Brazil nut (Bertholletia excels). J. Funct. Food 2010, 2, 196− 209. (25) Milbury, P. E.; Chen, C.-Y.; Dolnikowski, G. G.; Blumberg, J. B. Determination of flavonoids and phenolics and their distribution in almonds. J. Agric. Food Chem. 2006, 54, 5027−5033. (26) Monagas, M.; Garrido, I.; Lebrón-Aguilar, R.; Gómez-Cordovés, M. C.; Rybarczyk, A.; Amarowicz, R.; Bartolomé, B. Comparative flavan-3-ol profile and antioxidant capacity of roasted peanut, hazelnut, and almond skins. J. Agric. Food Chem. 2009, 57, 10590−10599. (27) Okuda, T.; Yoshida, T.; Hatano, T. New methods of analyzing tannins. J. Nat. Prod. 1989, 52, 1−31. (28) Lazarus, S. A.; Hammerstone, J. F.; Adamson, G. E.; Schmitz, H. H. High-performance liquid chromatography/mass spectrometry analysis of proanthocyanidins in food and beverages. Methods Enzymol. 2001, 335, 46−57. (29) Guyot, S.; Marnet, N.; Sanoner, P.; Drilleau, J.-F. Direct thiolysis on crude apple materials for high-performance liquid chromatography characterization and quantification of polyphenols in cider apple tissues and juices. Methods Enzymol. 2001, 335, 57−70. (30) Scioneaux, A. N.; Schmidt, M. A.; Moore, M. A.; Lindroth, R. L.; Wooley, S. C.; Hagerman, A. E. Qualitative variation in proanthocyanidin composition of Populus species and hybrids: Genetics is the key. J. Chem. Ecol. 2011, 37, 57−70. (31) Lea, A. G. H.; Bridle, P.; Timberlake, C. F.; Singleton, V. L. The procyanidins of white grapes and wines. Am. J. Enol. Vitic. 1979, 30, 289−300. (32) Rios, L. Y.; Bennett, R. N.; Lazarus, S. A.; Rémésy, C.; Scalbert, A.; Williamson, G. Cocoa procyanidins are stable during gastric transit in humans. Am. J. Clin. Nutr. 2002, 76, 1106−1110.
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Separation and Characterization of Phenolic Compounds from U.S. Pecans by Liquid Chromatography−Tandem Mass Spectrometry Katherine S. Robbins,† Yuanyuan Ma,† M. Lenny Wells,‡ Phillip Greenspan,§ and Ronald B. Pegg*,† †
Department of Food Science & Technology, College of Agricultural and Environmental Sciences, The University of Georgia, 100 Cedar Street, Athens, Georgia 30602-2610, United States ‡ Department of Horticulture, College of Agricultural and Environmental Sciences, The University of Georgia, 4604 Research Way, Tifton, Georgia 31793-5766, United States § Department of Pharmaceutical & Biomedical Sciences, College of Pharmacy, The University of Georgia, 250 W. Green Street, Athens, Georgia 30602, United States S Supporting Information *
ABSTRACT: The phenolic acids and proanthocyanidins (PACs) of pecans possess bioactive properties, which might be useful in retarding the onset of and ameliorating the status of certain chronic disease states. There is a general lack of information in the literature regarding such compounds, especially the PACs. Crude phenolic extracts pooled from eight commercially significant cultivars were selected based on their relatively high antioxidant capacities. The pooled extracts were separated via Sephadex LH20 column chromatography into five ethanolic low-molecular-weight (LMW) fractions and one acetonic high-molecular-weight (HMW) fraction. The preparations were then characterized using RP-HPLC-ESI-MS/MS and diol-phase HPLC-ESI-MS/MS in order to determine the key constituents present in the LMW and HMW fractions, respectively. As previously observed in pecan nutmeat, ellagic acid and (+)-catechin were found to be the major phenolics in the LMW fractions. The last eluting LMW fraction did not contain phenolic acids; rather it possessed PAC monomers and dimers. The HMW fraction comprised a majority of its PACs as dimers; yet, monomers, trimers, tetramers, pentamers, and hexamers were also separated and characterized. KEYWORDS: pecans, proanthocyanidins (PACs), Sephadex LH-20, RP-HPLC, diol-phase-HPLC, ESI-MS/MS
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INTRODUCTION Pecans [Carya illinoinensis (Wangenh.) K. Koch] and other tree nuts are receiving increased attention due to their purported health benefits1−4 and a qualified health claim approved more than 10 years ago by the U.S. Food and Drug Administration. Most reports associate these benefits with healthful lipid profiles (i.e., MUFAs, PUFAs, α-tocopherol, and γ-tocopherol), but more recently attention has turned toward the favorable phenolic profiles that tree nuts possess. Endogenous pecan phenolics can be segregated into two main classes, namely, phenolic acids (e.g., ellagic and gallic acids as well as their derivatives) and condensed tannins/proanthocyanidins (PACs) with varying degrees of polymerization (DP).5 Characterization of these important classes, especially the PACs, is lacking in the literature.6 PACs, or condensed tannins, are a type of phenolic polymer. There are two main types, that is, procyanidins and prodelphinidins. Procyanidins, the most prevalent PAC found in foods, are composed of (−)-epicatechin monomers, whereas the prodelphinidins comprise (−)-epigallocatechin monomers.7 PACs are usually not classified based on the individual compound type; rather, they are centered on the DP that exist.8 The DP can range anywhere from 2 (i.e., dimers) to 10+ (i.e., polymers). The antioxidant properties and antimutagenic activity of PACs can vary based upon structure and DP.7 Pecan PACs and their flavan-3-ol monomers have shown potential for important health-related benefits in vivo.6 In a randomized controlled trial employing a crossover design with © 2014 American Chemical Society
a one-week washout period between treatments, Hudthagosol et al.6 had 16 healthy men and women (23−44 years, BMI 22.7 ± 3.4) consume a test meal composed of either whole pecans, blended pecans, or an isocaloric meal of equivalent macronutrient composition. Blood was sampled at baseline and at intervals up to 24 h postingestion. Plasma antioxidant capacities and the levels of γ-tocopherol and epigallocatechin gallate (EGCG) from the panelists’ blood were determined; each increased significantly (p < 0.05) postprandially. While the flavan-3-ol monomers can be absorbed in the small intestine, there is evidence that all PACs with a DP of 3 and higher may not be able to be properly absorbed.9 If this is indeed the case, larger PACs can still participate in contributing a health benefit to humans, as these compounds can be broken down into smaller secondary metabolites by colonic microflora10 and then be absorbed. In this study, we employed Sephadex LH-20 column chromatography to fractionate pecan phenolic acids, their derivatives, and PACs. Once separated, these fractions were characterized by the total phenolics content (TPC) assay, UV spectral analysis, and RP-HPLC-ESI-MS/MS characterization of the low-molecular-weight (LMW) fractions and diol-phase Received: Revised: Accepted: Published: 4332
February April 11, April 15, April 16,
20, 2014 2014 2014 2014
dx.doi.org/10.1021/jf500909h | J. Agric. Food Chem. 2014, 62, 4332−4341
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analyzed. A total lipid and phenolic extraction was completed in triplicate for each of the 18 pecan cultivars. Sample Selection and Extract Separation. On the basis of preliminary data (results not shown here), the eight cultivars with the best performance based on the H-ORACFL assay were selected for use in this study. A 240 mg portion of each crude pecan extract was taken and pooled. The resultant extract (1.92 g) was mixed in a small volume of ∼75% (v/v) ethanol and sonicated to ensure its complete dissolution. The sample was then applied via a Pasteur pipet to the top of a chromatographic column packed with Sephadex LH-20 (bead size: 25−100 μm; Chromaflex column, 30 × 400 mm [i.d. × length], Kontes, Vineland, NJ, USA), and flow was conducted by gravity. The LMW phenolic compounds (molecular weight 10mers constituted 3.4, 8.5, 5.3, 20.5, 17.0, and 45.2%, respectively, in pecans. It should be noted that in the present study we did not find any PACs larger than hexamers. With the removal of the 7−10mers and >10 mers from the Gu et al.8 data set, the monomers, dimers, trimers, and 4−6mers now account for 9.2, 22.5, 13.9, 54.3%, respectively. In comparison to the prevalence of tetramers through hexamers in the former study, the present one found dimers to be most abundant. Earlier methods of PAC analysis (i.e., TLC analysis, RP-HPLC, and early normalphase HPLC methods) were discredited for failing to separate compounds with DP > 4.27 In preparing for this research, standards were run with DP of 8, 9, and even 10 with excellent resolution and detection on the diol-phase column and the fluorescence detector. For this reason, we believe that any PACs with DP > 7 would have been visible with this methodology and no suppression of ions occurred. In a study by Kelm et al.,15 in which the diol-phase HPLC methodology was first introduced, there was also excellent resolution up to a DP = 14. On the basis of Kelm et al.15 and the employment of the diol-phase column, we believe that PACs with higher DP would be properly resolved if they had been present in the defatted pecan acetonic extract. This was further confirmed by the MS data. No ions corresponding to PACs with a DP higher than 6 were identified in the sample. While the authors believe that all of the PACs containing (+)-catechin and (−)-epicatechin monomers were identified and quantified, there may be some PACs that are not properly captured with the fluorescence detector. Lazarus et al.28 points out that fluorescence detection cannot be used for the identification of PACs with gallocatechin and gallic ester components. Further HPLC investigation of these compounds using diol-phase diode-array detection is necessary. Additional information regarding specific composition, monomer position, and length of the PACs could be acquired using the thiolysis preparation technique coupled with HPLC characterization. In thiolysis, individual PAC units are separated into end units and interior units through an acid-catalyzed cleavage of the interflavan bonds.29 After cleavage, the interior unit intermediates are reacted with toluene α-thiol, forming thiol-flavonol adducts, and the end units remain as flavanol monomers.29,30 These thiol adducts can then be separated and identified with HPLC analysis to give a better picture of the average DP of the PACs, as well as the position and type of the monomeric units.30 We are not aware of any literature studies that have investigated pecan PACs using a thiolysis step. It has also been noted that when PACs are separated on Sephadex LH-20 via isocratic elution, the first compounds to elute will be those PACs with smaller MWs and the heavier ones will elute last.31 A possible reason for a reduction in the quantity of HMW PACs in our study could be that a sufficient quantity of the aqueous acetone was not employed to elute all PACs from the column. However, based on the mass balance performed in this work, it is not possible that this could account for the extreme differences observed. A total of 1.92 g of crude extract was loaded onto the Sephadex LH-20 column, and a mass of 1.90 g comprising the various fractions was recovered after lyophilization. This signifies that 99% of the crude extract applied to the column was recovered via the chromatography and that very small quantities of higher DP, if they existed, might remain on the Sephadex LH-20 column. Unfortunately the Gu et al.8 study is the only literature source that quantifies and characterizes PAC components of pecan nutmeat. Further
Table 4. Characterization and Quantification of the Tannin Fraction from a Pecan Crude Acetonic Extract Isolated by Sephadex LH-20 Column Chromatography Using 50% (v/v) Aqueous Acetonea degrees of polymerization (DP)
pooled cultivarsb
1 2 3 4−6 total
0.3 47.3 21.1 14.8 83.5
a
Characterized by diol-phase HPLC and quantification completed using commercial standards with varying degrees of polymerization. b All quantities are expressed as mg compound/g tannin fraction.
fraction(s). These findings demonstrate that the tannin fractions, in particular those with higher DP, may play a role in determining the observed in vitro antioxidant activity of a sample. A deeper examination of the PAC composition was completed using diol-phase HPLC-ESI-MS/MS analyses. After completing a full scan over the mass range of 200 to 3000 Da, a search of common PAC mass ions from the literature20,26 was undertaken: nine compounds were identified in the acetonic fraction (i.e., fraction VI; see Table 5). All of the Table 5. PAC Compounds Present in the Tannin Fraction Isolated from Eight Pooled Pecan Cultivars tRa
DPb
unit typesc
linkage
[M − H]−
6.5 9.7 11.0 13.1 14.2 16.0 16.3 18.0 19.0
2 3 3 4 4 5 5 6 6
2 (epi)catechin 3 (epi)catechin 2 (epi)catechin and 1 (epi)gallocatechin 4 (epi)catechin 3 (epi)catechin and 1 (epi)gallocatechin 5 (epi)catechin 4 (epi)catechin and 1 (epi)gallocatechin 5 (epi)catechin and 1 (epi)gallocatechin 4 (epi)catechin and 2 (epi)gallocatechin
B B B B B B B B B
577 865 881 1153 1169 1441 1457 1745 1761
a
Retention time determined from diol-phase HPLC-ESI-MS analyses. Degrees of polymerization. cCross-checking with compounds identified in nut skins, as reported by Sarnoski et al.20 and Monagas et al.26 b
compounds present in the sample exhibited a B-type linkage, which was to be expected based on literature reports of pecan PACs.8 Interestingly, the tannin fraction possessed both procyanidin and prodelphinidin compounds. Two PACs, one procyanidin and one prodelphinidin, were detected for each DP with the exception of the dimer, which had a single identification, and the hexamers, which were both prodelphinidins. The single dimer is composed of two (epi)catechin units based on a tR of 6.5 min and a molecular ion [M − H]− at m/z 577. The two trimers identified in the pecan sample had molecular ions [M − H]− at m/z 865 and 881 and eluted at a tR of 9.7 and 11.0 min, respectively. On the basis of the [M − H]−, these compounds were identified as possessing 3 (epi)catechin units and 2 (epi)catechin + 1 (epi)gallocatechin unit. The two detected trimers differed in their molecular ions by 16 amu, and this trend continued for the three larger DP of m/z at 1153−1169, 1441−1457, and 1745−1761. Gu et al.8 stated that PACs are present in numerous different food types including pecans. These authors reported that the 4339
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(2) Kris-Etherton, P. M.; Zhao, G.; Binkoski, A. E.; Coval, S. M.; Etherton, T. D. The effects of nuts on coronary heart disease risk. Nutr. Rev. 2001, 59, 103−111. (3) Blomhoff, R.; Carlsen, M. H.; Andersen, L. F.; Jacobs, D. R., Jr. Health benefits of nuts: potential role of antioxidants. Br. J. Nutr. 2006, 96, S52−S60. (4) Sabaté, J.; Ros, E.; Salas-Salvadó, J. Nuts: nutrition and health outcomes. Br. J. Nutr. 2006, 96, S1−S2. (5) Villarreal-Lozoya, J. E.; Lombardini, L.; Cisneros-Zevallos, L. Phytochemical constituents and antioxidant capacity of different pecan [Carya illinoinensis (Wangenh.) K. Koch] cultivars. Food Chem. 2007, 102, 1241−1249. (6) Hudthagosol, C.; Haddad, E. H.; McCarthy, K.; Wang, P.; Oda, K.; Sabaté, J. Pecans acutely increase plasma postprandial antioxidant capacity and catechins and decrease LDL oxidation in humans. J. Nutr. 2011, 141, 56−62. (7) Rigaud, J.; Escribano-Bailon, M. T.; Prieur, C.; Souquet, J.-M.; Cheynier, V. Normal-phase high-performance liquid chromatographic separation of procyanidins from cacao beans and grape seeds. J. Chromatogr. A 1993, 654, 255−260. (8) Gu, L.; Kelm, M. A.; Hammerstone, J. F.; Beecher, G.; Holden, J.; Haytowitz, D.; Gebhardt, S.; Prior, R. L. Concentrations of proanthocyanidins in common foods and estimations of normal consumption. J. Nutr. 2004, 134, 613−617. (9) Holt, R. R.; Lazarus, S. A.; Sullards, M. C.; Zhu, Q. Y.; Schramm, D. D.; Hammerstone, J. F.; Fraga, C. G.; Schmitz, H. H.; Keen, C. L. Procyanidin dimer B2 [epicatechin-(4β-8)-epicatechin] in human plasma after the consumption of a flavanol-rich cocoa. Am. J. Clin. Nutr. 2002, 76, 798−804. (10) Déprez, S.; Brezillon, C.; Rabot, S.; Philippe, C.; Mila, I.; Lapierre, C.; Scalbert, A. Polymeric proanthocyanidins are catabolized by human colonic microflora into low-molecular-weight phenolic acids. J. Nutr. 2000, 130, 2733−2738. (11) Wu, X.; Beecher, G. R.; Holden, J. M.; Haytowitz, D. B.; Gebhardt, S. E.; Prior, R. L. Lipophilic and hydrophilic antioxidant capacities of common foods in the United States. J. Agric. Food Chem. 2004, 52, 4026−4037. (12) Craft, B. D.; Kosińska, A.; Amarowicz, R.; Pegg, R. B. Antioxidant properties of extracts obtained from raw, dry-roasted, and oil-roasted US peanuts of commercial importance. Plant Foods Hum. Nutr. 2010, 65, 311−318. (13) Swain, T.; Hillis, W. E. The phenolic constituents of Prunus domestica. I.−The quantitative analysis of phenolic constituents. J. Sci. Food. Agric. 1959, 10, 63−68. (14) Srivastava, A.; Greenspan, P.; Hartle, D. K.; Hargrove, J. L.; Amarowicz, R.; Pegg, R. B. Antioxidant and anti-inflammatory activities of polyphenolics from southeastern U.S. range blackberry cultivars. J. Agric. Food Chem. 2010, 58, 6102−6109. (15) Kelm, M. A.; Johnson, J. C.; Robbins, R. J.; Hammerstone, J. F.; Schmitz, H. H. High-performance liquid chromatography separation and purification of cacao (Theobroma cacao L.) procyanidins according to degree of polymerization using a diol stationary phase. J. Agric. Food Chem. 2006, 54, 1571−1576. (16) Boulekbache-Makhlouf, L.; Meudec, E.; Chibane, M.; Mazauric, J.-P.; Slimani, S.; Henry, M.; Cheynier, V.; Madani, K. Analysis by high-performance liquid chromatography diode array detection mass spectrometry of phenolic compounds in fruit of Eucalyptus globulus cultivated in Algeria. J. Agric. Food Chem. 2010, 58, 12615−12624. (17) de la Rosa, L. A.; Alvarez-Parrilla, E.; Shahidi, F. Phenolic compounds and antioxidant activity of kernels and shells of Mexican pecan (Carya illinoinensis). J. Agric. Food Chem. 2011, 59, 152−162. (18) Mertz, C.; Cheynier, V.; Günata, Z.; Brat, P. Analysis of phenolic compounds in two blackberry species (Rubus glaucus and Rubus adenotrichus) by high-performance liquid chromatography with diode array detection and electrospray ion trap mass spectrometry. J. Agric. Food Chem. 2007, 55, 8616−8624. (19) Fracassetti, D.; Costa, C.; Moulay, L.; Tomás-Barberán, F. A. Ellagic acid derivatives, ellagitannins, proanthocyanidins and other phenolics, vitamin C and antioxidant capacity of two powder products
and more extensive characterization of these compounds is necessary to determine more information about the contents of pecans, in particular the HMW PACs. By utilizing exact mass HPLC/MS technologies, one might be able to further elucidate the structure of these compounds in pecans from various growing locations and cultivar types. HPLC/MSn analysis has previously been used to gain excellent information regarding the PAC profile of peanut skins.20 Researchers aim to quantify and characterize phenolic compounds in natural products due to their beneficial health benefits. The PAC compounds have been shown to remain intact in the low pH environment of the stomach and arrive to the small intestine intact.32 In this form, the PACs are unable to be absorbed in large quantities.10 However, colon microflora is able to breakdown PAC polymers to smaller metabolites that can be absorbed and utilized.10 The full benefit of these metabolites is not fully understood, thereby making predictions of the biological activity of higher degree polymers difficult. Hudthagosol et al.6 demonstrated adroitly that concentrations of the main PAC monomers, (+)-catechin and (−)-epicatechin, increased in the plasma of individuals after pecan consumption; these results are encouraging. Further research on the bioaccessibility (i.e., the extent of release of nutrients/ phytochemicals from the food matrix during digestion) and the bioavailability (i.e., the extent of absorption and utilization of released nutrients/phytochemicals for normal physiological functions) of phenolic constituents and PACs of pecans need to be investigated to gain better insight into the role of these compounds in disease prevention.
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ASSOCIATED CONTENT
S Supporting Information *
This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: 706-542-1099. Fax: 706-542-1050. E-mail: pegg@uga. edu. Funding
The authors would like to acknowledge the Georgia Agricultural Commodity Commission for Pecans (GACCP) and the USDA-NIFA-SCRI Award No. 2011-51181-30674 for funding this research. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Use of the Proteomics and Mass Spectrometry (PAMS) core facility under the direction of Dr. Dennis Phillips of UGA’s Department of Chemistry is greatly appreciated; in particular, thanks are extended to Dr. Phillips for assisting Y.M. with the LC-MS/MS analyses. The authors would also like to acknowledge Dr. Agnieszka Kosińska-Cagnazzo of the Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences for her assistance with setting up the diolphase HPLC analyses.
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REFERENCES
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