Almond Polyphenols: Methods of Analysis, Contribution to Food Quality, and Health Promotion Bradley W

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Almond Polyphenols: Methods of Analysis, Contribution to Food Quality, and Health Promotion Bradley W. Bolling

Abstract: Almond is a nutrient-dense tree nut recognized for its favorable lipid profile, vitamin E content, and polyphenols. The objectives of this review were to determine the polyphenols reported in almond, summarize the methods of analysis, and determine the polyphenol contribution to almond quality and health-promoting activity. Approximately 130 different polyphenols have been identified in almond, although not all of these have been quantitated. The mean and 25% to 75% percentile contents reported in literature were 162 mg (67.1 to 257) proanthocyanidins (dimers or larger), 82.1 mg (72.9 to 91.5) hydrolysable tannins, 61.2 mg (13.0 to 93.8) flavonoids (non-isoflavone), 5.5 mg (5.2 to 12) phenolic acids and aldehydes, and 0.7 mg (0.5 to 0.9) isoflavones, stilbenes, and lignans per 100 g almond. Following solvent extraction of almond, hydrolysis of the residue liberates additional proanthocyanidins, phenolic acids and aldehydes, and total phenols. Blanching and skin removal consistently reduces almond polyphenol content, but blanch water and almond skins retain enough polyphenols to be used as antimicrobial and antioxidant ingredients. Roasting and pasteurization have inconsistent effects on almond polyphenols. Almond polyphenols contribute to shelf life by inhibiting lipid oxidation and providing pigmentation, flavor, astringency, and antimicrobial activity. The health-promoting activity of whole almonds has been widely investigated, but few have considered the contribution of polyphenols. Preclinical studies of polyphenol-rich almond skin or almond extracts suggest putative effects on antioxidant function, detoxification, antiviral activity, anti-inflammatory function, and topical use for inhibiting ultraviolet A damage. Therefore, almond has a diverse polyphenol profile contributing to both its food quality and health-promoting actions. Keywords: almond, bioactivity, polyphenols, shelf life, tannins

Introduction Polyphenols contribute to almond quality. Almond phenols Tree nuts are a nutrient-dense food, perhaps best-known for are concentrated at the lipid interface to the environment, and having favorable fatty acid profiles. Tree nuts and their byprod- contribute to almond color, moderate astringency and enhance ucts also have significant polyphenol content (Chang and others shelf-life due to antioxidant and antimicrobial activity. Emerging 2016). Almond is among the most popular tree nuts produced evidence also suggests that polyphenols contribute to the health and consumed worldwide. Almond is consumed as the whole nut, benefits of almond consumption (Mandalari and others 2011a, but also may be blanched to remove its skin, roasted, processed to b). Furthermore, the cholesterol-lowering activity of nuts is not flours, nondairy beverages, or other ingredients for use in foods predicted entirely by unsaturated lipids. It was estimated that 25% and confections. of the nut cholesterol-lowering activity was due to components A growing body of evidence has associated nut consumption other than fatty acids (Kris-Etherton and others 1999). Almond to improvements in cardiovascular health, metabolic syndrome, polyphenols, phytosterols, and fiber may also contribute to lipid and diabetes (Ros 2015). Likewise, recent studies have demon- modulation. strated the cardiovascular benefits of almond consumption and Given the importance of polyphenols to almond quality, it is bioavailability of almond polyphenols (Garrido and others 2010; therefore important to accurately characterize almond polyphenol Berryman and others 2015; Grundy and others 2015; Jamshed and content and profile. Accurate analytical methods are necessary to others 2015; Ruisinger and others 2015). determine how polyphenols contribute to product quality and the health benefits of almond consumption. Therefore, the purpose of this review is to identify the almond polyphenols reported in literature, assess methods of identification and quantification, and CRF3-2016-0006 Submitted 12/16/2016, Accepted 2/8/2017. Author is to determine the contribution of almond polyphenols to product with Dept. of Food Science, Univ. of Wisconsin-Madison, 1605 Linden Dr., Madison, WI 53706, U.S.A. Direct inquiries to author Bolling (E-mail: quality and health benefits. In addition, almond polyphenol con- [email protected]). tent will be compared to other nuts and polyphenol-rich foods.

C 2017 Institute of Food Technologists® r 346 ComprehensiveReviewsinFoodScienceandFoodSafety Vol.16,2017 doi: 10.1111/1541-4337.12260 Almond polyphenols . . . ) LOD) Continued ( < (mg/100 g) Quantitative basis tannins, 1.28%) Skin (16.5 to 81.1) Blanched skin (qualitative) Almond (13.7 to 20.8) Almond skin extract (qualitative) Almond (3.96 to 10.7) Almond (qualitative) Blanched skin (16.5 to 37.0) Almond skin extract (qualitative) B3/B1, B2, B7, B5, C1,and A-type trimers, procyanidin K3R, dimers K3Glu, I3R, I3Glu,E7Glu,K,Q,I,DQ,N,Er Q3Glu, N7Glu, DP2-7, A and B-type linkages K3Glu, Er, Q, N, K, I cyanidin, Q, K, I, procyanidin B2 and B3 per 2009 study K3Glu, K3R, I3R, Er, Q, N, I B3/B1, B2, B7, B5, C1,and A-type trimers, procyanidin K3R, dimers K3Glu, I3R, I3Glu,E7Glu,K,Q,I,DQ,N,Er Q3Glu, N7Glu, Q3R, Q3Gal, DQ, Q3Glu, K3R,I3Glu/Gal, N7Glu, DK, I3R, Er, Q3R, Q, N, I, K Total flavonoidsTotal flavonoids Almond (500-2500) Hull extract (8468-23720) CA, EC, Q3Gal, N7GLu, Q3R, Q3Glu, DK, K3Gal, method Qualitative 3 3 Quantification ButanolysisMethanolysis Total tannins Hydrolyzable tannins Insoluble fiber (hydrolzyable Insoluble fiber (PACs HPLC-ESI-MS/MS CA, EC, procyanidins B1, B2, B3, B5, B7, C1 Almond (7.1 to 30.4) ButanolysisHPLC Total proanthocyanidins pHba, Va, Pa, P-a, Ca, Cga, CA, EC, procyanidins Skin (581 to 2880) MALDI-TOFHPLC-MS Propelargonidins,HPLC-MS procyanidins, prodelphinidins CA, EC, Q3R, N7R, Q3Gal, K3Gal, K3R, DK, I3R, I3Glu, Pa, pHba Almond (1.7 to 7.4) HPLC, Butanolysis Va, Ca, caffeic acid, ferulic acid, delphinidin, HPLC-MSHPLC-MSThiolysis Flavonoids/phenolic acids in 7 cultivars over 3 years Roasted/raw almonds, storage Monomers through tetracosamers of (epi)catechin Skin (qualitative) Almond skin HPLC pHba, Va, Pa, P-a, Ca, Cga, EC, CA, procyanidins C, 30 min, ° C, 45 min CE-ESI-TOF-MS pHba, Pa, Va Almond skin extract (qualitative) C, 45 min HPLC-ESI-TOF-MS Pa, Ca, pHba, Cga, Va Almond skin extract (qualitative) C, 45 min CE-ESI-TOF-MS Q3Glu/Gal, I3R, K3R, N7Glu, I3Glu/Gal, N Almond skin extract (qualitative) C, 45 min HPLC-ESI-TOF-MS CA, dihydrokaempferol-3-O-glucoside, EC, Er7Glu, C C ° ° ° ° ° ° C) ° C, 30 minC, 30 min MALDI MALDI I3R, I3Glu, K3R, K3Glu K, I, K3Glu, I3Glu, K3R, I3R Skin (7.5 to 24.5) Skin (9.3 to 9.5) ° ° C C ° ° C, 60 min AlCl ° C, 20 h C, 60 min AlCl ° ° blanching, Methanol/water/acetic acid blanching, Methanol/water/acetic acid 2 2 HCl–Butanol (1 h, 100 v/v), 85 acetone/water (70:30, v/v), sonication, 15 min methanol/water/acetic acid (50:48.25:1.75, v/v/v), 24 h, 4 methanol/water/acetic acid (50:48.25:1.75, v/v/v), 24 h, 4 sonicated for 15 min sonication for 15 min 5 min, followed by 15 min steeping acid hydrolysis (50:48.25:1.75, v/v/v), 24 h, 4 (50:48.25:1.75, v/v/v), 24 h, 4 methanol in water with 5%cycles) acetic acid (15 min, 3 5 min, followed by 15 min steeping Hydrolysable polyphenols: methanol/H2SO4 (10:1, Defat with n-pentane, extraction with Sequential ASE extraction, or Sequential ASE extraction, or Hexane, 70% methanol (reflux) 60 LN 70% methanol, 23 70% methanol, 23 Aqueous, methanol, or acetone extractionBlanch/roast, methanol/HCl (1000:1, v/v) and Blanch/roast, methanol/HCl (1000:1, v/v) and Butanolysis Total proanthocyanidins Skin (15 to 4880) Water, 25 10 mL of methanol/HCl (1000:1, v/v) by sonication, LN Defat, acetone/water (80:20, v/v), 50 Hexane, 70% methanol (reflux) 60 ASE, subsequent extractions of 90, 60, 30% 10 mL of methanol/HCl (1000:1, v/v) by sonication, Hexane, 70% methanol (reflux) 60 Hexane, 70% methanol (reflux) 60 Methanol, 25 ´ an and e´ and others ´ aez-Rom ˜ ni and others 2009 Condensed tannins: isolate fiber, 5 mL/L 2013 2009 others 2010 2010 2002 Sporns 2002 2007 2008 2010 2011a 2005 1988 2008 2010 2008 Go Bittner and others Bolling and others Arr Bolling and others Frison and Sporns Frison-Norrie and Garrido and others Garrido and others Barriera and others Bartolom Bolling and others Table 1–Studies reporting almond polyphenols and method of analysis. ReferenceAmarowicz and others Extraction method Brieskorn and Betz Chen and Blumberg Barreira and others

r C 2017 Institute of Food Technologists® Vol.16,2017 ComprehensiveReviewsinFoodScienceandFoodSafety 347 Almond polyphenols . . . ) Continued ( (mg/100 g) Quantitative basis vanillin; 2945 CE by vanillin, 0.41 polymerization index) highest with PEG400 mixture. lowest with methanol, highest with 40% PEG200 solution Skin (qualitative) Skin (25 to 234) Almond (2.84 to 5.92) Skin (693 to 944) Skin (qualitative) Skin (167 to 204) Almond (qualitative) Almond (184) Almond (5.2 to 6.9) Almond extract (ND-517) catechin > > - ,4 catechin > -dihydroflavonyl-4 10 ,3 > diferulate (benzofuran form) -methoxy-2 oligomers Q3Glu, K3R, N7Glu, I3R, K3Glu, I3Glu, Er, Q, N, K, I 8,5 gallate K3Glu, Er, N, Q, I, K oxymethyl-5,7-dimethoxyflavone (biflavone) (epi)catechin, and (epi)catechin glycoside, DPpolymers,avg.DP12.7or8.5,including 1 to monomers; Terminal units: epicatechin Extension units: epicatechin epiafzelechin through DP I3R, K3R, N7Glu, K, N, I K3Glu, I3Glu, Er, Q, N, K, I CA, EC, predominately dimers and trimers, with Tannins present Almond (qualitative) Total flavonoids Almond extract (ND-545 mg CE) B-type proanthocyanidins with (epi)afzelechin 3 complexion Total chlorogenic acid Almond skin (200 to 1250), 3 method Qualitative ) 3 Quantification HPLC-MS precipitation HPLC-MS/MS HPLC Ga, Pa, 5Hba, Cga, Va, Ca Almond extract (142 to 1269) HPLC-UV/FL, Total flavonoids, AlCl HPLC-MS Secoisolariciresinol, biochanin A, genistein Almond (0.084) HPLCHPLC, HPLC-MS/MS CA, EC, Q3R, N7Glu, I3R, Q3Gal, K3R, Q3Glu, I3Glu, CA, cyanidin, EC, epicatechin gallate, gallocatechin HPLC-FLHPLC, Procyanidin HPLC-MS/MS and propelargonidin type monomers pHba, CA, EC, Q3R, Q3Glu, I3Glu, K3Glu, Q, I3Glu, Q, HPLCModified vanillin assayAl(NO Total proanthocyanidins CA, EC, Er7Glu, Q3R, Q3Gal, Q3Glu, K3R, N7Glu, I3R, Almond skin (200 to 2500) Normal phase C, C, C, ° ° ° C, 10 min. The ° C or defatted in C UPLC-MS/MS Phenolic acids, flavonoids Almond (11 to 42) ° ° C, 24 h ° C ° 30 min (70:29.5:0.5), C18 SPE clean-up 30 min min, 4 to 6 enzymatic hydrolysis methanol and 1.2 N HCl with TBHQ hexane, methanol/water/acetic acid (50:48.25:1.75, v/v/v), 37 vortexed for 30 s, sonication at 37 Sephadex clean up 30 min min, room temperature, varying PEG solution/pH min, room temperature, varying solvents,solution/pH PEG tube was inverted once into the suspend middle the of samples. sonication Roommin; temperature, Sephadex 50 LH-20 isolation Defat by hexane, ethanol/water (80:20, v/v), 80 Defat with hexane, acetone/water/acetic acid Methanol with 0.1% HCl, sonication 15 minMethanol with 0.1% HCl, sonication 15Hydrolysis min of cell-wall material HPLC HPLC Pa, HPLC 5Hba, CA, Cga, Va, EC, Ca, Er7Glu, Q3R, Q3Gal, 5Hba, Cga, Pa, Ca, Va pHba, Va, pHb-a, Ca, Fa, 5,5’-diferulate, Blanch water (0.2 mg/100 mL) Defat by hexane, methanol/water (52:48, v/v), 26 Blanching 30 to 600 s, 25 or 100 10% methanol in sodium acetate (0.1%, pH 5), Hot water blanching Hydrolysis or vanillin Total proanthocyanidins Skin extract (7184 CyE, by After in vitro digestion HPLC Total flavan-3-ols, flavanols, flavanones Skin (400 to 3460) Acetone, water (80/20, v/v), 50 90% methanol with TBHQ or Acid hydrolysis with Acetone/water (80:20, v/v), Sephadex clean-up Modified vanillin assay Condensed tannins present Almond (qualitative) Defat by hexane, ethanol/water (80:20, v/v), 80 Methanol with 0.1% HCl, sonication 15 min HPLC Pa, 5Hba, Cga, Va, Ca Skin (3.43 to 23.7) Acetone/water/acetic acid, 70:29.5:0.5 v/v/v; Blanch skin, drying, sonication 30 min, 120 W, 15 ¨ uven and ¨ Ozg 1999 2010c (Mandalari and others 2010a,d) 2008 2012 2008 2013 2010b others 2015 2006 ¨ ultekin- Lin and others 2016 Defat by hexane, ethanol/water (80:20, v/v), 80 Lazarus and others Mandalari and others Hughey and others Hughey and others Karamac 2009Kuhnle and others Ethanol Vanillin, protein Mandalari and others Mandalari and others Table 1–Continued. ReferenceGu and others 2003 Acetone, water, acetic acid, 70/29.5/0.5, v/v/vGu and others 2004G HPLC-MSHarnly Acetone, and water, others acetic acid, 70/29.5/0.5, v/v/v; Extraction method Epicatechin glycosideJoshi and others 1987 Ethanol Almond (qualitative) NMR 4; Ma and others 2014 Blanch skin, drying, sonication 30 min, 120 W, 15

r 348 ComprehensiveReviewsinFoodScienceandFoodSafety Vol.16,2017 C 2017 Institute of Food Technologists® Almond polyphenols . . . ) Continued ( (mg/100 g) Quantitative basis 3.03%; tannins: 0.36% to 1.87%) Almond (qualitative) Blanch water extract (qualitative) Skin (qualitative) Skin (qualitative) Blanch water extract (89.1) Skin (polyphenols: 0.13% to Almond (0.894) Hulls (qualitative) Skin (9.3 to 10.6) Skin (9.3 to 23.3) Unripe fruit (qualitative) Almond (16 to 27) )-sesamin, + B1, B2, B7, B6, C1, A-type + )-pinoresinol, )-lariciresinol, + + )-syringaresinol, 7-hydroxysecoisolariciresinol, )-matairesinol, ( )-socoisolariciresinol, + − − )-7-Hydroxymatairesinol, ( glucoside, (epi)catechin trimer, catechin, (epi)afzelchin-(epi)catechin dimer, (epi)catechin-ethyl trimer, EGlu, KR, IR,Er,Q,K,I KGlu/Gal, containing A-/B-type linkages, (epi)afzelechin, (epi)catechin, (epi)gallocatechin, galloylation heptamers containing (epi)catechin, (epi)afzelechin, and (epi)gallocatechin (epi)catechin, (epi)gallocatechin units and tetramer (1). B- andProcyanidins, A- prodelphinidins, type and dimers. propelargonidins. butanolysis ( cyclolariciresinol, ( ( ( secoisolariciresinol-sesquilignan cinnamic acid derivative, epigallocatechin gallate, CA, EC, I3R or rhamnetin,or proanthocyanidins, methoxyhesperetin Q3R rhamnoside, glycosylated rhamnetin DQ,N,Er dimers, A-type trimers dimers and trimers), small amountdimers of A-type DK, K3Gal, I3Gal, K3Glu, K3R, I3R, E, Q, N, K, I − method Qualitative , Butanolysis Polyphenols as catechin-equivalents; tannins after 4 Quantification HPLC-MS/MSHPLC-MS CA dihexoside, CA hexoside, (epi)catechin dimer, DQ hydroxybenzoic acid derivatives, vanilloylhexose, MALDI-TOF MS At least 52 unique proanthocyanidins through DP10 HPLC-MS/MS ( HPLC, HPLC-MSMALDI-TOF-MSMALDI CA, EC, procyanidins B3 A/B-type linkages from monomers through HPLC-MS/MS Procyanidin trimers to octamers containing 20 unique proanthocyanidin dimers (14), trimers (5), HPLC, HPLC-MSHPLC, HPLC-MS pHba, Va, Pa, P-a, Ca, Cga K3R, K3Glu, I3R, I3Glu, Q3Glu, N7Glu, Er7Glu, K, Q, I HPLC-MS Skin Procyanidins (4.2 B3, to B1, 8.4) B4, B2, B7, C1, B5, C2 (15 HPLC-ECD CA, Pa, EC, pHBA, Va, Q3Gal, N7Glu, Q3R, Q3Glu, C, Sephadex ° 20 C, 30 min, Sephadex C, 30 min HPLC-FL, HPLC-MS CA, EC, procyanidin B2 Skin (49.3) ° ° − C, 16 h ° C, 24 h HPLC-MS, NMR I3Glu, I3Gal, I3R, N7Glu, K3R Skin (qualitative) ° C, 90 min, subsequent ethyl acetate ° subsequent partitioning extraction after suspension in water with methanol/water (50:50, v/v; pHtemperature, 2), 1 room h, then acetone/waterv/v) (70:30, enzymatic hydrolysis, acid/base hydrolysis sonication for 15 min twice sonication for 15 min twice clean-up with methanol/water (50:50, v/v; pHtemperature, 2), 1 room h, then acetone/waterv/v) (70:30, (50:46.3:3.7 v/v/v), 4 sonication for 15 min twice sonication for 15 min twice LH-20 Ethanol, 25 Defat, ethanol/water (70:30, v/v) 24 h, triplicate, 80% acetone (1:10, w/v), 50 80% acetone (1:10, w/v), 50 Industrial blanch water, water soluble or extracted 0.1 M HCl in methanol, reflux, 2 hASE, defat, acetone, 70% acetone in water (v/w), GC-MS pHba, Va, Pa, Ga Skin (0.1) Ethyl acetate, ethanol, or water KMnO Blanched skin, methanol/HCl (1000:1, v/v) Blanched skin, methanol/HCl (1000:1, v/v) Methanol, solication 15 min, Industrial blanch water, water soluble or extracted Methanol/water (70:30), sonicationHot-water blanching, methanol/water/HCl Methanol/HClBlanched skin, methanol/HCl (1000:1, v/v) Blanched skin, methanol/HCl (1000:1, v/v) HPLC Phenolic acids, flavonols, flavan-3-ols, flavanones HPLC Skin (22.6) Ga, ellagic acid after hydrolysis Almond (not detected) enez´ and 2007 2016 2009 1983 2007 others 1983 others 1998 Torres 2012 2016 2006 2008 2007 erez-Jim ´ Rubilar and others Qureshi and others Monagas and others Senter and others Smeds and others Song and others 2010 Not reported Not reported Pa, EC, Q, N, K Almond (1.2) Skin (0.4) Sang and others 2002Saura-Calixto and 95% ethanol, 50 de Pascual-Teresa and P Table 1–Continued. ReferenceMandalari and others Milbury and others Molyneux and others Monagas and others Extraction method

r C 2017 Institute of Food Technologists® Vol.16,2017 ComprehensiveReviewsinFoodScienceandFoodSafety 349 Almond polyphenols . . . u, g) μ (mg/100 g) )-catechin; DQ, dihydroquercetin; + Quantitative basis bound: 4.03), Skin extract (free: 1.63; bound: 27.8), Almond (qualitative) with 70% ethanol, blanched almond skins 0.018 isoflavones) vanillin) hull extract (free: 0.14; bound: 96.5) bound: 3.4 to 6.4) ellagic acid: 48.7 to 63.2) 93.5 total) Almond (70 to 290) Skin extract (123) Almond extract (qualitative) Almond (0.131 phytoestrogens, Almond (0.112) Skin (30.8 to 33.2) Skin (1.45 to 3.62) Hulls [79.5:14.8:5.7] )-epicatechin; CA, ( − oxyresveratrol Almond (1.19 to 8.52 + -caffeoylquinic acid (neochlorogenic O -caffeoylquinic acid (cryptochlorogenic O methanol. unknown B-type propelargonidin dimer, procyanidin B7, unknown A-type propelargonidin dimer, proyanidin C1 K3R, N, N7Glu, unknown A-typedimer propelargonidin glycitein secoisolariciresinol K3Glu, I3R, I3Glu, quercetin, kaempferol, isorhamnetin, dihydroquercetin, eriodictyol, N7Glu, naringenin acid, chlorogenic acid acid), 3- acid) Catechin, epicatechin, Q3R, Q3Glu, QGal, K3R, Protocatechuic acid, p-hydroxybenzoic acid, vanillic DP 4.2, predominately (epi)catechin, A-type dimer Skin extract (24% of extract by Free, bound, total flavonoids Almond (39.8 free, 53.7 bound, lyethylene glycol; TLC, thin layer chromatography; ECD, electrochemical detection; EC, ( maric acid; Q, quercetin; pHba, p-hydroxybenozic acid; P-a, protocatechuic aldehyde; Ga, gallic acid; pHb-a, p-hydroxybenzaldehyde. e; K, kaempferol; K3Gal, kaempferol-3-O-galactoside; K3Glu, kaempferol-3-O-glucoside; K3R, kaempferol-3-O-runtinoside; N, naringenin; N7Gl method Qualitative 3 Quantification HPLC-MS HPLC-MS phloroglucinolysis, HPLC-MS HPLC-MS/MS CA, EC, K3R, I3Glu, I3R, Q3R, N3Glu, N Skin (46 to 116.2). Max recovery HPLC-FL/UV-MSHPLC-FLD Procyanidin B2, unknown A-type procyanidin dimers, HPLC-UV-MS EC, CA, DQ, Er, Er7Glu, I, I3Gal, I3R, K, Ellagic K3Gal, acid, K3Glu, Pa, Va Commercial skin extract (0.51) GC-MSMALDI-TOF/MS Dimers to 11mers Lariciresinol, matairesinol, pinoresinol, Skin (qualitative) HPLC-UV/FL, AlCl HPLC-UV/FL, HPLC-FLHPLC, TLCUPLC-MS Proanthocyandins by degree of polymerization Hydrolyzable tannins as gallic acid, ellagic acid Polydatin, piceatannol Almond: (free: 25.4 to 107.3; Almond: (gallic acid: 14.1 to 36.8; Butanolysis, vanillin, HPLC DAD Ga, CA, Cga, caffeic acid, EC, Ca, Q, K Almond (104.25) C, 30 ° C, derivatization GC-MS Coumesterol, daidzein, formonenetin, genistein, ° C, Alkaline hydrolysis, ° C for 30 min HPLC Pa, Q3R, K3Glu, morin, K3R, I3Glu, Q, I Almond, almond skin, almond hull ° C, 24 h ° ethanol/water (70:30, v/v) (70:30, v/v), 40 extracted with ethyl acetate extraction with ethyl acetate extracted with ethyl acetate enzymatic hydrolysis, derivatization (52:48, v/v) hydrolysis (52:48, v/v) Sephadex clean-up Sephadex clean-up, methanolysis enzymatic hydrolysis, SPE clean-up h, Toyopearl TSK HW-40-F clean-up min Defat, 80% ethanol, 80 Defat, 80% ethanol, reflux, hydrolysis HPLC Caffeic acid, Fa, Ca, sinapic acid Almond extract (free: trace, Methanol, acidified methanol,Methanol or methanol/HCl (100:1, v/v) Vanillin assay Vanillin/nonspecific Tannins present Total tannins, 3.14-fold increase with acidified Almond (qualitative) Microwave-assisted extraction (MAE), stirring, Methanol, ethanol, acetone, or acetonitrile/water water/HCl (1000:1, v/v) and sonicated for 15 min, Water, HCl (1000:1, v/v), sonicated 15 min, water/HCl (1000:1, v/v) and sonicated for 15 min, Defatted, 70% methanol, 60 to 70 70% methanol, 60 to 70 Methanol/HCl (1000:1, v/v), Methanol/water Diethyl ether, acetone/water (80:20, v/v), 50 Acetone/water/acetic acid (70:29.5:0.5, v/v/v), Diethyl ether, methanol by Soxhlet extractionAcetone/water (70:30, v/v), room temperature, 48 TLC, HPLC Cga, 4- 2006a 2006b Sathe 2006 2015 2013 2009; Also reported in: (Llorach and others 2010) 2006 2010 2003 2014 Wijeratne and others Wijeratne and others Venkatachalam and Valdes and others Tsujita and others Urpi-Sarda and others Thompson and others Yang and others 2009 80% acetone, with hexane wash or alkaline Teets and others 2009 Methanol/HCl (1000:1, v/v), Methanol/water Xie and others 2012Xie Acetone/water/acetic and acid Bolling (70:29.5:0.5, 2014 v/v/v), Ethanol–water homogenization, extraction, Yildirim and others Table 1–Continued. ReferenceTakeoka and Dao Truong and others Extraction method CE, capillary electrophoresis; ASE, acceleratedEr, solvent extraction; eriodictyol; FL, fluorescence; Er7Glu, TOF,naringenin-7-O-glucoside; time-of eridictyol-7-O-glucoside; Q, flight; quercetin; I, TBHQ, Pa, tert-butylhydroquinone; protocatechuic isorhamnetin; PEG, acid; po 5Hba, I3Gal, 5-hydroxybenzoic acid; isorhamnetin-3-O-galactoside; Cga, I3R, chlorogenic acid; isorhamnetin-3-O-rutinosid Va, Vanillic acid; Ca, cou

r 350 ComprehensiveReviewsinFoodScienceandFoodSafety Vol.16,2017 C 2017 Institute of Food Technologists® Almond polyphenols . . .

Methods galloylation of almond proanthocyanidins has not been reported. Peer-reviewed primary literature on almond polyphenols were The degree of galloylation is important, as it affects molar absorp- retrieved by searching CAplus database, indexing more than 1500 tivity and reduces the fluorescent response, which could lead to journals in the area of chemistry, food science, and biology using an underestimation of total proanthocyanidin content by HPLC SciFinder (American Chemical Society) and PubMed.gov that in- analysis. cludes MEDLINE database. References from recent reviews and Almond proanthocyanidins interflavan bonding appears to be book chapters including almond polyphenols we also evaluated mainly B-type, having carbon–carbon bonds at C4→C6 or (Bolling and others 2011b; Fallico and others 2011; Alasalvar C4→C8 (Gu and others 2003). Almonds also have A-type carbon and Bolling 2015; Chang and others 2016). After an exhaus- bonding, although the configuration of these bonds have yet to tive literature search, 61 peer-reviewed studies reporting qualita- be identified (Figure 2). A-type bonds are either C2→C7 and tive or quantitative data on almond polyphenols were identified with either C4→ C6 or C4→ C8, or C2→ C5 with C4→ C6 (Table 1). Data from these studies were aggregated and reported bonds (Figure 1). Although both A- and B- type bonds have been as mg equivalents per 100 g almond or 100 g almond skin. reported, the proportion of these bond types is not known. Almond proanthocyanidin dimers (procyanidin B1, B2, B3, B5, Identification and Quantitation of Almond B7) have been identified and quantitated by HPLC-MS methods Polyphenols by comparison to authentic standards (Figure 2; de Pascual-Teresa Polyphenols identified and quantitated in almonds and others 1998; Monagas and others 2007). A-type dimers, B- Polyphenols have been characterized from extracts of whole type oligomers, and mixed B-/A- type oligomers up to DP 11 almond, almond blanch water, almond skins, and almond hulls. have been identified by MALDI analysis (Monagas and others Based on these reports, data were compiled and summarized. In- 2007, 2009; Perez-Jim´ enez´ and Torres 2012; Tsujita and others dependent of almond type (roasted, raw, cultivar, or year), the 2013). Nearly 70 unique proanthocyanidins were identified in most abundant polyphenols were proanthocyanidins, hydrolysable almond blanch water (Perez-Jim´ enez´ and Torres 2012). However, tannins, and flavonoids, with 61 to 162 mg/100 g almonds proanthocyanidins larger than DP 11 are present, as the average (Table 2 and A1). Lesser amounts of phenolic acids, lignans, DP of almonds proanthocyanidins was found to be 12.7 (Gu and isoflavones, and stilbenes were reported (0.7 to 5.5 mg/100 g others 2003). The majority of almond proanthocyanidins appear almonds). The sum of almond polyphenols was 312 mg/100 g to be polymers by normal-phase HPLC analysis (Figure 2). almond. Less polyphenols have been reported in almond skin, The intrinsic complexity and diversity of almond proantho- 90 mg/100 g skin, likely because the majority of reports uti- cyanidins, as well as a lack of available standards poses analytical lized hot water-blanched skin as the starting material (Table 2 and challenges. The solubility of proanthocyanidins in aqueous and or- A2). Blanching depletes almond skin polyphenols, thus it is not ganic solutions is limited, which may limit extractability. Typically, possible to infer content from this data to a whole almond ba- acetone/water/acetic acid (70:29.5:0.5, w/w/w) or varying pro- sis (Milbury and others 2006). Almond hull extracts are also rich portions of acidified aqueous acetone have been solvents of choice sources of polyphenols and have similar polyphenols to whole al- for extracting proanthocyanidins (Gu and others 2003; Garrido mond (Takeoka and Dao 2003; Wijeratne and others 2006a, b; and others 2007). However, a small proportion of almond proan- Rubilar and others 2007; Barriera and others 2010). Although thocyanidins are not extractable by acetone/water, and liberated almond hull is not presently used as a source of food ingredients, after alkaline hydrolysis. Unextractable almond proanthocyanidins developing an almond hull extract could increase the value of this varied by cultivar, and ranged from 3.4 to 6.4 mg/100 g among waste stream (Esfahlan and others 2010). Nonpareil, Butte, and Carmel almond (Xie and others 2012). Also, The methods of identification of almond polyphenols were ultrasonic extraction of blanched almond skins with polyethylene mainly by high-performance liquid chromatography (HPLC) cou- glycol (PEG) yielded >65% more proanthocyanidins than ace- pled to UV/VIS detection and mass spectrometry. In some cases, tone/water extraction (Ma and others 2014). Thus, it appears that matrix-assisted laser desorption/ionization (MALDI) mass spec- prior methods may have underestimated almond proanthocyanidin trometry (MS), gas chromatography-MS (GC-MS), or nuclear content. magnetic resonance (NMR) analysis has been used to iden- Furthermore, quantitation of almond proanthocyanidins by tify structures (Senter and others 1983; Joshi and others 1987; normal-phase HPLC were based on (+)-catechin equivalents or Monagas and others 2007; Perez-Jim´ enez´ and Torres 2012). standards isolated from cocoa and blueberry to quantitate almond proanthocyanidins (Gu and others 2004; Xie and others 2012). Almond proanthocyanidins In contrast to almonds, blueberry and cocoa are only B-type, do Proanthocyanidins are flavan-3-ol monomers, oligomers, and not contain (epi)afzelechin, and are not galloylated (Gu and others polymers that form anthocyanidins upon hydrolysis in mineral 2003). Thus, the accuracy of these measurements require fur- acids (Ferreira and others 1999). For this review, flavan-3-ol ther validation. Isolation and purification of an authentic almond monomers will be categorized with flavonoids, and proantho- proanthocyanidin standard or fraction would be expected to im- cyanidins categorized as dimers or higher degree of polymeriza- prove quantitative analysis by HPLC, as well nonspecific methods, tion (DP). By this classification, proanthocyanidins are the most such as reaction with vanillin or 4-dimethylaminocinnimaldehyde abundant class of polyphenols in almonds, although only 2 quan- (DMAC; Krueger and others 2013). Furthermore, it appears that titative studies report proanthocyanidins in whole almond and are normal-phase HPLC analysis is complicated by the presence of hy- indexed in nutrient databanks (Table 2 and A3). drolysable tannins, which co-elute by Sephadex LH-20 clean-up Almond proanthocyanidins consist of mainly (−)-epicatechin (Xie and others 2012). and (+)-catechin, with lesser amounts of (−)-epiafzelechin A number of analytical strategies are available to characterize (Figure 1; Gu and others 2003). Further, these units may also proanthocyanidins (Table 3; Neilson and others 2016). Butanoly- be galloylated or polygalloylated (Monagas and others 2007; sis or DMAC assays provide semiquantitative data on polyphe- Perez-Jim´ enez´ and Torres 2012). The quantitative proportion of nol content. MALDI analysis provides data on the degree of

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Table 2–Polyphenol proﬁle of almonds and almond skins compiled from literature reports.a

Mean, rangeb (mg/100 g) Polyphenol class Whole almond Almond skins Proanthocyanidins (dimers and larger) 162 (67.1–257) 6.98 (4.16–9.70) Hydrolysable tannins 82.1 (72.9–91.5) – Flavonoids, non-isoﬂavones 61.2 (13.0–93.8) 71.3 (18.7–104) Phenolic acids and aldehydes 5.5 (5.16–12.2) 11.6 (3.83–18.9) Minor phenolic (isoﬂavones, stilbenes, lignans) 0.7 (0.5–0.9) – Sum of classes 312 (161–450) 90.0 (29.5–132) aData are means of quantitative data reported in literature, as mg/100 g almond or mg/100 g almond skin. –, not reported in literature. bRange is the 25% to 75% percentile of individual compounds.

Table 3–Analytical methods to characterize dietary proanthocyanidins.

Method DP A or B bonding Cis or trans bonding Branching Flavan-3-ol type Quantitative HPLC-UV/FLD Xa Xa NMR X X X X MALDI X X X X MALS X X ESI X X X Xa Thiolysis Xa XX XXa Phloroglucinolysis Xa XXXXa Polarimetry X SAXS X X X X DMAC Xa Butanolysis Xa Alkaline hydrolysis prior to HPLC Xa Xa

MALs, multiangle light scattering; ESI, electro-spray ionization; SAXS, small-angle X-ray scattering; DMAC, 4-dimethylaminocinnamaldehyde. aLimited or incomplete data obtained by method. polymerization, A- or B-type bonding, and flavan-3-ol composi- proanthocyanidin and flavan-3-ol content of almonds, it is possible tion, but is not quantitative (Reed and others 2005). Normal-phase that cyanidin was an artifact of the extraction process. For exam- HPLC provides information about the degree of polymerization, ple, delphinidin and cyanidin were recovered after acid hydrolysis and is semiquantitative. NMR, thiolysis, phloroglucinolysis, or of almond extract (Amarowicz and others 2005). Other studies polarimetry can provide further information about the degree of have not identified anthocyanidins in almonds, almond skin, or cis-ortrans-interflavan bonds. Thus, combinations of methods almond blanch water. are needed to fully characterize proanthocyanidin composition of Almond flavan-3-ols include (+)-catechin, dihydrokaempferol, foods. The cis–trans configuration, A-/B-type ratios, and flavan- dihydroquercetin, (-)-epicatechin, epicatechin gallate, epicatechin 3-ol types of almond proanthocyanidins have not been adequately glycoside, and gallocatechin gallate. Dihydrokaempferol, (+)- characterized. catechin, and (-)-epicatechin were the most abundant flavan-3- ols, having an average of 2 to 4 mg/100 g almond. (+)-Catechin Hydrolysable tannins and (-)-epicatechin have been most frequently identified in al- Hydrolysable tannins include gallotannins, ellagitannins, and mond and almond skin (Table A1 and A2). Epicatechin gallate and phlrotannins, which release gallic acid, ellagic acid, and phloroglu- epicatechin glycoside have been identified in almond, but have not cinol upon hydrolysis (Figure 3). Ellagitannins and gallotannins yet been quantified (Gu and others 2003; Harnly and others 2006). have been identified in almond after hydrolysis, with ranges of Flavonols were the most abundant flavonoid class in almond and 53 to 57 and 20 to 34 mg/100 g, respectively (Xie and others include isorhamnetin, kaempferol, quercetin and their 3-O-glu- 2012). Ellagic acid was also identified and quantified in a com- cosides, galactosides, and rutinosides. Morin has also been iden- mercial almond skin extract at 0.51 mg/100 g, which had not been tified, but not quantified in almond skin (Wijeratne and others hydrolyzed (Urpi-Sarda and others 2009). The parent structures 2006a). Similarly, isorhamnetin-3-O-galactoside was identified in of almond hydrolysable tannins are not yet characterized. Pre- almond skin, but has only been quantitated in whole almond liminary analysis by thin-layer chromatography indicated at least (Arraez-Rom´ an´ and others 2010). The relative almond flavonol 5 to 6 different gallotannins and 6 different ellagitannins with profile was that isorhamnetin-3-O-rutinoside > isorhamnetin- large molecular weights (Xie and others 2012). Relative to other 3-O-glucoside kaempferol-3-O-rutinoside > other flavonols polyphenol classes, this class has few reports, particularly given its (Figure 4). However, in almond skins, quercetin-3-O-rutinoside abundance in almonds. was more abundant than isorhamnetin-3-O-glucoside (Figure 4). The flavanones eriodictyol, naringenin, and their 7-O- Almond flavonoids glucosides have been identified in almond and almond skins. At least 25 different flavonoids have been identified in almonds Naringenin and naringenin-7-O-glucosides accounted for the or its coproducts (Figure 4 and 5). Anthocyanidins, flavan-3- majority of flavanone content, at 3 mg/100 g almond. ols, flavonols, flavanones, and a biflavone have been identified in Eridicytol-7-O-glucoside has been identified in almond skin and blanch water, but not quantitated in whole almond. In ad- almond, almond skins, or almond blanch water (Figure 4). Cyanidin, the lone almond anthocyanidin, was identified after dition, the biflavone 4; -methoxy-2 ,3 -dihydroflavonyl-4 ,4 - acid hydrolysis of almond (Harnly and others 2006). Anthocyani- oxymethyl-5,7-dimethoxyflavone was isolated from almond skins dins, such as cyanidins can be formed from hydrolysis of proan- and identified using NMR and mass spectrometry (Joshi and oth- thocyanidins (Amarowicz and others 2005). Given the significant ers 1987).

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Figure 1–Structures of representative proanthocyanidins reported in almonds. The absolute stereochemistry of A-type proanthocyanidin dimer, epicatechin-afzechin, and epiafzelechin dimers have not been conﬁrmed. Proanthocyanidin with degree of polymerization of more than 10 have been reported, and more than 70 different proanthocyanidins have been identiﬁed in almond or almond blanch water (Perez-Jim´ enez´ and Torres 2012).

Phenolic acids and aldehydes sinapic acid, and 2 diferulates have been identified in almond skin, Almond phenolic acids and aldehydes have been identified by although only chlorogenic acid, caffeic acid, and p-coumaric acid HPLC-MS, capillary electrophoresis (CE)-MS, and GC-MS anal- have been quantitated. Almonds may have other hydroxycinnamic ysis. Almond skin phenolic acids and aldehydes have been better acids, as hulls contain 4-O-caffeoylquinic acid (cryptochlorogenic characterized than from whole almond (Figure 6 and 7). The acid) and 3-O-caffeoylquinic acid (neochlorogenic acid; Takeoka hydroxybenzoic acids p-hydroxybenzoic acid, 5-hydroxybenzoic and Dao 2003). acid, vanillic acid, and protocatechuic acid have been identified Similar to other polyphenols, phenolic acids are known to be as- and quantitated in almond skin, with a mean of 8.31 mg/100 g. sociated with cell wall material and portion inaccessible by solvent Hydroxybenzaldehydes have been identified and quantitated in al- extraction. Almond cell wall material had 2 mg phenolic acids mond skin, but not quantitated in whole almond. Almond skin and aldehydes per gram, which included p-hydroxybenzoic acid, protocatechuic acids ranged from 0.5 to 2.0 mg/100 g. The hy- vanillic acid, p-hydroxybenzaldehyde, vanillin, p-coumaric acid, droxycinnamic acids chlorogenic acid, ferulic acid, caffeic acid, ferulic acid, and diferulic acids (Mandalari and others 2010c).

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Figure 2–Content of almond (A) tannins, (B) proanthocyanidins, and (C) proanthocyanidins (skins) reported in literature. Bars represent ranges and means. Bars at origin signify quantitative data not present in literature. For (A) data were derived from Gu and others (2004) and Xie and others (2012), where proanthocyanidins determined by normal phase HPLC analysis. These tannins were not reported blanch water or almond skins. For C, ∗ indicates this compound detected in blanch water or blanch water extract. Additional proanthocyanidins reported by Perez-Jimenez and others (2012).

Most quantitative reports utilized methanol/water/acid extrac- Lignans tions for phenolic acids and aldehydes. HPLC-MS or CE-MS Lignans are a minor polyphenol class present in almonds, analysis assess flavonoids and phenolic acids in the same method having 674 μg/100 g (Figure 8 and 9). The lignans profile (Monagas and others 2007; Hughey and others 2008; Bolling and was such that (−)-socoisolariciresinol > (+)-lariciresinol > others 2009; Teetsand others 2009). Based on the chromatography cyclolariciresinol (+)-pinoresinol > (+)-sesamin > (+)- reported in these studies, it is expected that pursuing ultra-high syringaresinol > (−)-7-hydroxymatairesinol > (−)-matairesinol pressure liquid chromatography (UHPLC) methods would im- 7-hydroxysecoisolariciresinol. Compounds were identified and prove separation, reduce analysis time, and permit identification quantitated by GC-MS or HPLC-MS/MS analysis (Thompson of additional flavonoids and phenolic acids. Also, the extraction and others 2006; Kuhnle and others 2008). In both cases, efficiency can be improved upon current methods. Sonication of compounds were isolated after hydrolytic methods. Therefore, it almond skin with PEG/water increased chlorogenic acid recovery is not clear if these compounds exist as conjugates or glycosides by 200% compared to methanol extraction (Ma and others 2014). in almonds.

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Figure 3–Hydrolysable tannins yield gallic acid or ellagic acid. Although almonds tannin fractions yield ellagic acid and gallic acid after hydrolysis (Xie and others 2012), although the parent compounds are not yet identiﬁed.

Isoflavones and coumesterol piceatannol and/or oxyresveratrol but were detected directly from Isoflavones were the least-abundant flavonoid subclass in al- almond blanch water. Similar to other polyphenols, stilbenes monds, having 38 μg/100 g almond (Figure 8 and 9). Biochanin were concentrated in skins, and readily extracted by hot water A is was the most abundant at 25 μg/100 g, with lesser amounts blanching. of genistein, daidzein, glycitein, and formonenetin. Daidzein, formonenetin, genistein, glycitein, and coumesterol were identified Total phenols by GC-MS, following derivatization (Thompson and others 2006; The Folin–Ciocalteu method is a nonspecific colorimetric de- Figure 8). Biochanin A and genistein were detected after extraction termination of “total phenols.” The assay is based on electron in methanol/sodium acetate (10:90, v/v) and enzymatic hydrolysis reduction of heteropolyphosphotungstates-molybdates, forming a (Kuhnle and others 2008). Thus, it is possible that biochanin A and blue pigment (Singleton and others 1999). Most methods report genistein may be present as glycosides or other parent compounds total phenols values as gallic acid equivalents (GAE), although oth- in almond. ersmayuse(+)-catechin, quercetin, or other polyphenols. Pheno- lics do not react equally in this assay, so the choice of the polyphe- Stilbenes nol equivalent will affect quantitation. For example, querecetin Almond stilbenes were reported in whole almond, meat, and is more than twice as reactive as gallic acid (Everette and others blanch water (Figure 8 and 9; Xie and Bolling 2014). Polydatin 2010). The food matrix, as well as individual components (amino (resveratrol-3-O-glucoside) was identified in Butte, Carmel, and acids, sugars, and others) can also create inconsistencies in the assay Nonpareil almonds at 7.19 to 8.52 μg/100 g. However, pteros- (Singleton and others 1999; Bolling and others 2012). tilbene and resveratrol were not detected. Solid-phase extrac- A wide range of value for total phenols have been reported tion (SPE) clean-up of almond extract did not sufficiently retain from extracts of whole almond, almond skin (blanched and

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Figure 4–Flavonoid content reported in almond skins. Bars represent ranges and means. Bars at origin signify quantitative data not present in literature. Data derived from references in Table 1. For B, ∗ indicates compound was detected in blanch water or blanch water extract. Compounds quantitated in blanch water [mg/100 mL] included (+)-catechin [0.23], (−)-epicatechin [0.13], eriodictyol-7-O-glucoside [0.5–1.1], naringenin [0.01], naringenin-7-O-glucoside [0.3], isorhamnetin-3-O-glucoside [5.4], isorhamnetin-3-O-rutinoside [0.2], kaempferol-3-O-glucoside [0.3], quercetin-3-O-glucoside [0.002], and quercetin-3-O-rutinoside [0.03].

Table 4–Total phenols values reported for almonds. 8 California almond cultivars. The variability of 16 flavonoids and 2 phenolic acids among Nonpareil, Butte, Carmel, Sonora, Material Value (per 100 g) Range N Fritz, Mission, and Monterey almond cultivars was measured Whole almond 190 mg GAE 47–1340 21 1193 CE 1010–1393 4 over 3 harvest seasons (Bolling and others 2011a). The mean Almond skin 1603 mg GAE 140–3471 22 polyphenol content varied from 6.1 to 7.0 mg/100 g over 3 4016 QE 0.369–11900 14 y in Butte, Carmel, and Nonpareil almonds. (−)-Epicatechin Skinless kernel 67 mg GAE 64–71 8 Oil 14.6 mg GAE 12.4–16.8 2 content changed the most between seasons, nearly 1.7-fold between 2005 and 2007 harvests. The intercultivar differences were GAE, gallic acid equivalents; CE, catechin equivalents; QE, quercetin equivalents. more pronounced, as content varied from 4 to 11 mg/100 g. Although polyphenol profiles were similar, Butte had rela- natural), skinless kernels, and oil (Table 4). Extraction of almond tively more kaempferol-3-O-rutinoside and isorhamnetin-3-O- rutinoside, Mission had increased flavanones, and Nonpareil skin by aqueous acetone (80:20, v/v; 50:50; v/v, or acidified + acetone) resulted in higher total phenols values than aqueous and Sonora had increased isorhamnetin-3-O-glucoside and ( )- methanol (Garrido and others 2007). Microwave-assisted extrac- catechin. Variability in flavonoid and phenolic acid content was tion also enhanced recovery of total phenols from almond skin also examined in 14 Turkish almond genotypes (Yildirim and oth- (Valdes and others 2015). Alkaline hydrolysis of almond after sol- ers 2010). Similar to California almonds, Turkish almond genotypes also had significant variability of flavonoids. For example, vent extraction approximately doubles the recovery of almond + total phenols (Yang and others 2009; Vinson and Cai 2012). ( )-catechin varied from 1.1 to 22.7 mg/100 g, and quercetin varied from 0.08 to 3.6 mg/100 g. Other studies have reported differences between California to Spanish almond skins (Mona- Variability of Almond Polyphenol Content gas and others 2007; Garrido and others 2008). Valdes and oth- Preharvest factors ers compared the flavonoid content of Spanish (Marcona, Guara, The impact of almond genotype and harvest year on polyphe- Planeta) and California (Butte, Colony, Carmel, Padre) almond nol content has been assessed by several groups. Milbury and skins (Valdes and others 2015). Notably, Guara and Padre were rich others (2006) reported 16 to 27 mg flavonoids/100 g among in kaempferol-3-O-rutinoside and isorhamnetin-3-O-rutinoside,

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Figure 5–Flavonoids reported in whole almond, almond skins, or almond blanchwater. whereas Planeta was rich in quercetin-3-O-rutinoside, but not Postharvest factors isorhamnetin-3-O-rutinoside. Thus, almond cultivars have unique Blanching, roasting, pasteurization, irradiation, and storage polyphenol profiles. The extent these differences affect specific al- can affect almond polyphenols. Polyphenols are concentrated mond quality parameters (for example, shelf-life, oxidation, flavor, in almond skins and are readily lost by hot water blanching color) have not been reported. (Milbury and others 2006). Thus, almond blanch water is also Among tannins, Butte, Carmel, and Nonpareil had similar mean rich in polyphenols (Milbury and others 2006; Bolling and oth- value of gallotannins and ellagitannins (Xie and others 2012). ers 2009; Perez-Jim´ enez´ and Torres 2012; Mandalari and others However, samples ranged from 14.1 to 40.6 mg/100 g gallotannins 2013). A significant number of studies that have characterized and 48.7 to 63.2 mg/100 g ellagitannins. Almond proanthocyani- polyphenols in almond skins, have utilized dried, blanched skin— din content had a higher degree of variability than hydrolysable a byproduct of the almond industry. Thus, these values cannot be tannins. Butte, Carmel, and Nonpareil almond had 32.2 to 111 mg extrapolated to whole almonds. Lab-scale liquid-nitrogen blanch- proanthocyanidins/100 g (Xie and others 2012). Blanched Span- ing has been utilized to isolate polyphenol-rich almond skins for ish and U.S. almond skin also had significant differences in use in nutrient accessibility studies (Mandalari and others 2010d; proanthocyanidin dimers and trimers, having 10.6 and 4.1 mg/ Grundy and others 2016). 100 g, respectively (Monagas and others 2007). Given these dif- Few studies have examined the effect of pasteurization on ferences, it would be expected that DP, bonding, and other as- almond polyphenols. Flavonoids and phenolic acids of almond pects of proanthocyanidin structures would vary between almond pasteurized by different methods did not significantly change genotypes. by processing method (Bolling and others 2010). However,

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Figure 6–Phenolic acids and aldehydes reported in almond skins. Bars represent ranges and means. Bars at origin signify quantitative data not present in literature. Data derived from references in Table 1. + indicates detected in whole almond or almond skin. For B, ∗ indicates compound detected in blanch water. Compounds quantitated in blanch water [mg/100 mL] included 5-hydroxybenzoic acid [0.2], protocatechuic acid [0.04], vanillic acid [0.02], 3-O-caffeoylquinic acid (chlorogenic acid) [0.04], trans-p-coumaric acid [0.004]. irradiation of almond skin powder at doses from 10 to 30 kGy nol content only increased in skins from whole almonds. Skins increased extractable polyphenols, possibly by disrupting cell removed by liquid-nitrogen blanching did not have increased structure and increasing polyphenol extractability (Teets and polyphenol content during accelerated aging. This suggests a com- others 2009). plex interplay between component of the whole almond and skin Roasting disrupts almond cell wall structures (Altan and others are necessary for developing increased polyphenols. 2011; Grundy and others 2016) and can affect almond polyphenols. Commercial roasted almond had similar levels of flavonoid Polyphenol Extraction Methods and phenolic acids as raw almonds (Bolling and others 2010). The choice extraction method is likely the most important However, roasted almond total phenols by the Folin assay were ap- factor in the recovery of almond polyphenols during analysis. proximately 25% less than raw almond (Bolling and others 2010). Almond polyphenols have been extracted from different matrixes, A recent study subjected almonds to various roasting conditions including whole raw/roasted almonds, almond skins, and blanch and assessed phenolic and flavonoid content (Lin and others 2016). water. Polyphenol extraction is typically a liquid–solid extraction Apparently, the roasting process is bi-phasic, where flavonoid and from whole almonds consists of grinding, defatting with hexane or phenolic acids decrease within 5 to 10 min of roasting, but increase another nonpolar solvent to remove lipids. Almond skins have been at 20 min. Roasting 20 min at 200 °C led to a higher recovery extracted by similar methods, although are not typically defatted. of flavonoids and phenolic acids compared to roasting at 150 °C. Defatted almond or almond skin powder is then subjected to solid– A similar trend was apparent for proanthocyanidins, as quantitated liquid extraction. Almond blanch water has been extracted by by a nonspecific vanillin assay. Although the mechanism remains phase partitioning, or drying blanch water and subsequent solid– to be identified, competing degradation and increased extractabil- liquid extraction. ity likely explain increased content at advanced stages of roasting. Few studies have reported systematic approaches to almond Roasted almond skins are also a byproduct of processing, and the polyphenol extraction. Solid–liquid extractions are affected by flavan-3-ols and phenolic acids have been profiled (Garrido and time, temperature, pressure, solvent, and particle size, agita- others 2008; Monagas and others 2009). Notably, skins isolated by tion, and solid–liquid ratio. Almond extraction has been per- industrial roasting had higher phenolics than hot-water blanching formed in a batch or continuous extraction. Batch extraction (Garrido and others 2008). (methanol/water/acetic acid, 50:48.25:1.75, v/v/v, 4 °C, 8 to Storage of raw almonds for up to 15 mo at 4 and 23 °C increased 10 h, twice) yielded similar flavonoids and phenolic acids as a flavonoid and phenolic acid content in almond skins (Bolling and continuous extraction by assisted solvent extraction (ASE; 100 °C, others 2011). Storage increased both almond p-hydroxybenzoic 1500 psi, 80%, 60%, 30% methanol in 5% acetic acid for 3 cycles acid and flavonols. In the same study, accelerated aging with in- of 15 min each; Bolling and others 2009). However, recovery of creased temperature and humidity also increased almond polyphe- flavonoid aglycones were higher from liquid-nitrogen blanched nols Interestingly, under accelerated aging conditions, polyphe- skin at the expense of flavonoid glycosides. This difference

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Figure 7–Phenolic acids and aldehydes reported in almond, almond skin, or almond blanch water. was not observed in hot-water blanched skins. Further, total re- nonpolarity (Hughey and others 2012). Also, 73% of the pheno- covery of flavonoids and phenolic acids was improved by hot-water lics were extracted within 2 min of hot water blanching, and 90% blanching and subsequent extraction. recovered after 10 min blanching (Hughey and others 2012). Others have compared the recovery of total phenols and Extraction conditions have not been optimized for hydrolysable proanthocyandins using water, methanol/water/HCl, and ace- tannins. Although ellagic acid after hydrolysis was reported in tone/water/HCl (Garrido and others 2007). Methanol/HCl whole almonds (Xie and others 2012), ellagic acid was not de- (1000:1, v/v) was the most effective solvent for recovery of tected by others (Molyneux and others 2008; Abe and others polyphenols from almond skin and shells. Ma and others (2014) 2010). Hydrolysable tannins are present in the proanthocyanidin- compared the proanthocyanidin and chlorogenic acid extraction containing Sephadex LH-20 fraction employed by many groups efficiency of PEG, acetone, methanol, and ethanol by ultrasound- as a clean-up step during analysis (Xie and others 2012). assisted extraction. Maximum recovery was obtained with 120 W, Thus, developing extraction and clean-up methods to detect 1:20 w/v, and PEG/water (50:50, v/v), relative to other sol- and quantitate almond hydrolysable tannins is expected to be a vent/water combinations. challenging task. Blanching time and temperature also affects the recovery of Methanol/water/acid systems are the most commonly em- almond polyphenols (Hughey and others 2012). Higher tem- ployed for extraction of almond flavonoids and proanthocyanidins, perature and time increased recovery of (+)-catechin and (−)- although acetone/water/acetic acid has been used for proantho- epicatechin, but room temperature increased overall extraction cyanidins. Based on the results by Ma and others (2014), the use of efficiency compared to 100 °C (Hughey and others 2012). solvent that improves polyphenol extractability may lead to better Polyphenol aglycones precipitated from blanch water due to their characterization of polyphenol content.

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Figure 8–Content of minor polyphenols reported in almonds. Bars represent ranges and means values. Data are from references in Table 1. +, detected in whole almond; ∗, detected in almond skin and blanch water.

Almond Polyphenol Functionality lowing irradiation than peeled almond flour (Lanza and others Polyphenol contribution to almond quality 2013). However, the extent to which application of additional Antioxidant activity. Almond polyphenols have well- polyphenols to almonds, or the differences in shelf-life between al- documented antioxidant activity (Amarowicz and others 2005; mond cultivars with varying polyphenol profiles have not yet been Barreira and others 2008; Garrido and others 2008). A tannin-rich explored. fraction isolated from almond extract inhibited oxidation in a Almond color. Almond polyphenols also significantly con- β-carotene-linoleate model system (Amarowicz and others 2005). tribute to the color of the seed coat. Almond flavonoids have yel- A similar lipid system identified distinct differences in antioxidant low pigmentation, but the brown almond skin pigment is largely activity between almond cultivars (Barreira and others 2008). concentrated in the high-molecular weight fraction, and can be Thus, almond polyphenols are expected to improve stability of purified using Sephadex LH-20 (unpublished observation). This lipids within almonds and also in bulk oil systems (Wijeratne and pigmentation contributes to the acceptability of almond (Larrauri others 2006b). and others 2016). Almond polyphenols may also contribute to Antimicrobial activity. Almonds skin also has antimicrobial the degree of browning developed during roasting, as polyphenols properties which may arise from synergistic interactions between may appear to affect Maillard browning in other foods (Noor- flavonoids and phenolic acids (Mandalari and others 2010a). Al- Soffalina and others 2009). Maillard browning and lipid oxidation mond skin extracts inhibited the pathogens Salmonella enterica, Lis- also contribute to intense browning of the almond kernel after teria monocytogenes, and Staphlococcus aurus (Mandalari and others heating, known as concealed damage (Rogel-Castillo and others 2010a). Almond extracts from liquid-nitrogen blanched skin also 2015). Postharvest moisture exposure appears to be the main fac- inhibited Escherichia coli, Serratia marcescens, Streptococcus mutans tor for the development of concealed damage (Rogel-Castillo and (Smeriglio and others 2016). others 2015). Shelf life. Almond shelf-life is determined primarily by lipid Almond flavor and aroma. Almond aroma is a complex mix- oxidation. Therefore, antioxidant almond polyphenols are ex- ture of products resulting from lipid autoxidation and degra- pected to contribute to the shelf-life of almonds. For example, dation and sugar degradation and the Maillard reaction in the almonds coated with carboxymethyl cellulose and peanut skin case of roasted almonds (Hojjati and others 2016; Erten and extract had reduced oxidation in roasted almonds (Larrauri and Cadwallader 2017). The characteristic mild astringency of al- others 2016). Unpeeled almond powder had less off-odors fol- mond skin is derived from its polyphenols, most likely tannins

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Figure 9–Minor polyphenols reported in almond, almond skin, or almond blanch water. and flavonoids given their abundance. Reports of the relative as- try. However, further development and optimization of industrial- tringency of almond polyphenols are presently lacking. scale methods could increase innovation in this area. Almond polyphenols as functional natural preservatives. Al- mond polyphenols have antioxidant and antimicrobial function, which is appealing as a clean-label natural food preservative. For Contribution of almond polyphenols to health example, the addition of almond skin powder reduced lipid oxi- A large body of evidence suggests that consumption of whole dation produces in raw minced chicken breasts during refrigerated almonds reduces cardiovascular disease risk by modulating plasma and frozen storage (Teets and Were 2008). Almond skin pow- lipoproteins (Berryman and others 2011). Almond consump- der also reduced oxidation and extended shelf life of ground beef tion also increases diet quality and contributes to satiety (Chen (Prasetyo and others 2007). Other than meats, almond polyphe- and others 2015; Hull and others 2015). Lipids from whole al- nols may be considered as preservatives for products containing monds have in delayed bioaccessibility, reducing net caloric utiliza- nuts or other products prone to lipid oxidation (Wijeratne and tion and reduced postprandial lipemia (Grundy and others 2015, others 2006b). Given the interest of utilizing natural preserva- 2016). Furthermore, emerging evidence suggests whole almond tives, almond polyphenols appear to be a promising source of consumption may be also beneficial for reducing inflammation, either antioxidant or antimicrobial activity. At this time, almond oxidative stress, and dysmetabolism occurring in metabolic syn- polyphenols are not widely used as antioxidants in the food indus- drome and type 2 diabetes (Kamil and Chen 2012).

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Almonds and other nuts contribute to polyphenol intake and Blumberg 2008). Almond skin polyphenols protected 3- (Saura-Calixto and others 2007; Zamora-Ros and others 2013). dimensional skin cultures from UVA radiation damage (Evans- Almond polyphenols are bioavailable and extensively biotrans- Johnson and others 2013). Liquid-nitrogen blanched almond skin formed by the microbiota and host tissue upon consumption (Gar- inhibited replication of herpes simplex virus type 2 in human pe- rido and others 2010). The precise function of almond polyphenol ripheral blood mononuclear cells (Arena and others 2015). Con- metabolites in prevention of chronic disease is still emerging. Uri- sumption of polyphenol-rich almond skin has also benefited im- nary excretion of urolithin A glucuronide, an ellagitannin metabo- mune function in rodent models of disease by reducing the effect lite, was associated with reduced symptoms of metabolic syndrome of spinal cord injury (Mandalari and others 2011b) and chemical- after 12 wk consumption of mixed nuts (Tulipani and others 2011, induced colitis (Mandalari and others 2011a). These peripheral 2012; Mora-Cubillos and others 2015). Also, almond polyphenols functions suggest almond polyphenols could contribute to detox- may broadly impact the metabolic state. For example, healthy mice ification and cancer prevention, prevention of cellular stress, and that consumed a polyphenol-rich almond extract had significant act as immunomodulators. changes in the metabolomic profile representing plasma amino acid metabolism and urinary nutrient and redox metabolism (Jove Almond Polyphenol Content Compared to Other Nuts and others 2011). The polyphenol content of tree nuts has recently been reviewed The determination of almond polyphenol content is expected (Alasalvar and Bolling 2015; Chang and others 2016). Other tree to inform nutritional epidemiology and intervention studies nuts also have phenolic acid, flavonols, flavones, flavanols, antho- (Zamora-Ros 2016). Recent methods have attempted to char- cyanins, isoflavones, condensed and hydrolysable tannins (Alasalvar acterize the polyphenol metabolome (Achaintre and others 2016; and Shahidi 2009; Bolling and others 2011b). Considering these Zamora-Ros and others 2016). The contribution of almond to the reports, almonds have moderate total polyphenol content relative polyphenol metabolic pool is unknown, but the pool is expected to other nuts. to be dominated by proanthocyanidin and catechin metabolites, A number of studies have compared the total phenol content which increases after almond consumption (Bartolome´ and others of nuts by the Folin–Ciocalteu assay. Chestnut, walnut, pecan, 2010; Garrido and others 2010; Zamora-Ros and others 2016). and pistachio have consistently higher total phenols than almond Prebiotics. Almond polyphenols are closely associated with (Chang and others 2016). Kornsteiner and others (2006) extracted fiber, and cell walls remain intact after simulated digestion of al- tree nuts and peanuts with acetone/0.526 mM sodium metabisul- mond skins (Mandalari and others 2010d). The close association of fite (75:25, v/v), which is expected to preferentially extract tan- polyphenols with fiber increases polyphenol delivery to the colon nins. Almonds had 239 mg GAE/100 g (ranging from 130 to 456), (Saura-Calixto and others 2007). Almond skins also increase pro- similar mean content to hazelnut. Pecans, peanuts with skin, pis- biotic bacteria in culture, including bifidobacteria and eubacteria tachios, and walnuts had 420 to 1625 mg GAE/100 g. Krings and (Mandalari 2009). Consumption of whole almonds did not in- Berger (2001) reported more than 50% less polyphenols in almond crease bifidobacteria after consuming 42.5 or 85 g/d for 2 wk ethanolic extract compared to hazelnut. Abe and others (2010) (n = 18) (Ukhanova and others 2014). However, the number of reported lower total phenols values from roasted almond (1114 butyrate-producing microbiota increased over this time, suggest- mg GAE/100 g), also similar to roasted hazelnuts and Brazil nut. ing a prebiotic effect (Ukhanova and others 2014). Thus, almond Vinson and Cai (2012) determined 18% or 27% of the phenolic polyphenols may contribute to gut health through modulation of content was bound in roasted and raw almonds by the Folin assay. gut microbiota. Given the present analysis, almondis among the richest sources Antioxidant function. The effect of almonds on antioxidant of nut flavonoids, having 61 mg/100 g compared to the 65 mg/ function has been previously reviewed (Bolling and others 2011b). 100 g or less reported in other nuts (Alasalvar and Bolling 2015). Almond polyphenols inhibited ex vivo cholesterol oxidation Almond stilbene content is similar or lower than peanut and pis- (Chen and others 2005). Likewise, almond consumption inhibited tachio (Alasalvar and Bolling 2015). Walnut, pecan, and chestnut biomarkers of oxidative stress in former smokers, older hyperlipi- have higher levels of ellagitannins (Alasalvar and Bolling 2015). demic adults, and Chinese adults with type 2 diabetes (Li and Hazelnut, pecan, and pistachio have higher proanthocyanidin con- others 2007; Jenkins and others 2008; Liu and others 2013). Al- tent than almonds. Only chestnut has higher reported hydrolysable mond consumption increased plasma α-tocopherol content (Chen tannin content than almond, although cashew and pecan levels ap- and other 2015), so the extent polyphenols contribute to an- pear similar (Alasalvar and Bolling 2015). tioxidant consumption from whole almonds is presently unclear. Given the limits of polyphenol bioavailability and the extensive Almond Polyphenol Content Compared to Other Foods catabolism and phase 2 metabolism that reduces antioxidant activ- Almond was ranked number 40 among the foods indexed ity, it is unlikely that almond polyphenols provide a direct antioxi- in phenol-explorer database (Perez-Jimenez and others 2010). dant function in vivo. However, it is plausible that that polyphenol Among the top 40 polyphenol rich foods, 12 were spices, which intake can confer protection through upregulation of antioxidant serving sizes are expected to be much less than 100 g. Given the function. present analysis, almonds would be within the top 26 polyphenol- Other known bioactivities. Almond polyphenols inhibited α- rich foods, having 312 mg/100 g. amylase activity, and its consumption reduced blood glucose in Foods with comparable polyphenol content in the phenol- rats (Tsujita and others 2013). Almond skin extract also inhibited explorer database (within 50 mg/100 g) included milk choco- rat primary hepatocytes against hydroperoxide and dicarbonyl- late, strawberry, red chicory, red raspberry, coffee, dried ginger, induced stress (Dong and others 2010). Similarly, almond skin whole grain hard wheat flour, prune, black grape, red onion, green proanthocyanidin-rich extract induced phase 2 detoxification en- chicory, fresh thyme, refined maize flour, soy tempeh, whole grain zymes and reduced acetaminophen-induced toxicity in mice after rye flour, and apple (Perez-Jimenez and others 2010). consumption (Truong and others 2014). Almond skin polyphe- Almond compares favorably to commonly consumed fruits and nols increased quinone reductase activity in hepa1c1c7 cells (Chen vegetables in the U.S. diet (Table 5). Almond polyphenol content

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Table 5–Mean content of almond polyphenols compared to other commonly consumed fruits.

Food PACs Polyphenols (non-PAC) Subclasses Total phenols (Folin) Almond 162 150 Flavan-3-ol, flavonol, hydrolysable tannin, isoflavone, lignin, phenolic acid 287 (127–418) Apple 109 56.3 Anthocyanin, dihydrochalcone, flavonol, phenolic acid 201 (66–430) Banana 4 2.5 Flavanol, phenolic acid 155 (12–231) Strawberry 145 97.5 Phenolic acid, anthocyanin, flavanol 289 (73–443) Potato – 28.3 Hydroxycinnamic acid 70 (17–163) Lettuce – 8.0 Flavone, flavonol, phenolic acid) 66 (4–131) Onions (white) – 5.4 flavonol) 45 Tomato – 4.2 Flavanone, flavonol, phenolic acid 45 (24–96) Orange juice – 65.4 Flavanone, flavones, flavonol 49 (36–76) Blueberry (highbush) 180 310 Anthocyanin, flavanol, Flavonol, phenolic acid 223 (20–868)

Data from Phenol-Explorer (Rothwell and others 2013), except for almond PACs and polyphenols which are from Table 1. PACs, proanthocyanidins; –, not reported. is similar to apple and blueberry, although it is not rich in antho- cyanins. Consumption of 1.5 oz of almonds (42 g) would supply References 131 mg of phenolics. Estimates of polyphenol intake range from Abe LT, Lajolo FM, Genovese MI. 2010. Comparison of phenol content and antioxidant capacity of nuts. Cienc Tecnol Alimen 30:254–59. 5 mg to 1 g/d so almonds contribute a reasonable amount of Achaintre D, Bulete A, Cren-Olive C, Li L, Rinaldi S, Scalbert A. 2016. dietary polyphenols from a single serving. Differential isotope labeling of 38 dietary polyphenols and their Ellagitannins intake mainly occurs from strawberries, raspberries quantification in urine by liquid chromatography electrospray ionization and blackberries, at <5 mg/d (Clifford and Scalbert 2000). In the tandem mass spectrometry. Anal Chem 88:2637–44. Phenol-Explorer database, only walnut and chestnut have higher Alasalvar C, Shahidi F. 2009. Natural antioxidants in tree nuts. Eur J Lipid Sci Technol 111:1056–62. ellagic acid or ellagic acid after hydrolysis content (Rothwell and Alasalvar C, Bolling BW. 2015. Review of nut phytochemicals, fat-soluble others 2013). Thus, almonds could be a significant source of ellag- bioactives, antioxidant components and health effects. Br J Nutr 113 Suppl itannin in the diet. Among gallic acid or gallotannin-containing 2:S68–78. foods reported in Phenol-Explorer, only raw chicory, cloves, and Altan A, McCarthy KL, Tikekar R, McCarthy MJ, Nitin N. 2011. Image chestnut have higher content than almonds. analysis of microstructural changes in almond cotyledon as a result of processing. J Food Sci 76:E212–21. Amarowicz R, Troszyn´ska A, Shahidi F. 2005. Antioxidant activity of Conclusions almond seed extract and its fractions. J Food Lipids 12:344–58. Polyphenols make an important contribution to almond qual- Arena A, Bisignano C, Stassi G, Filocamo A, Mandalari G. 2015. Almond ity. Almond polyphenols are mainly composed of tannins and skin inhibits HSV-2 rReplication in peripheral blood mononuclear cells by flavonoids, with lesser levels of phenolic acids and aldehydes, modulating the cytokine network. Molecules 20:8816–22. lignans, and stilbenes. Tannins and flavonoids are expected to Arraez-Rom´ an´ D, Fu S, Sawalha SMS, Segura-Carretero A, contribute the most significantly to the functionality of almond Fernandez-Guti´ errez´ A. 2010. HPLC/CE-ESI-TOF-MS methods for the characterization of polyphenols in almond-skin extracts. Electrophoresis polyphenols, but the synergistic or additive actions of minor phe- 31:2289–96. nolics should not be overlooked. Given the limitations of prior Barreira J, Ferreira I, Oliveira M, Pereira J. 2008. Antioxidant activity and analytical approaches and the complexity of almond polyphenol bioactive compounds of ten Portuguese regional and commercial almond biosynthesis, it is likely that further investigations will identify cultivars. Food Chem Toxicol 46:2230–35. previously uncharacterized polyphenols. Thus, this complex- Barreira J, Ferreira I, Oliveira M, Pereira J. 2010 . Anitoxidant potential of chestnut (Castanea sativa L.) and almond (Prunus dulcis L.) by-products. ity provides a considerable challenge in developing standardized Food Sci Tech Inter 16:0209–8. methods of polyphenol analysis. Innovative approaches to quan- Bartolome´ B, Monagas M, Garrido I, Gomez-Cordov´ es´ C, Mart´ın-Alvarez´ titation are needed to account for incomplete resolution of phe- PJ, Lebron-Aguilar´ R, Urp´ı-SardaM,LlorachR,Andr` es-Lacueva´ C. 2010. nolics, particularly tannins. Identifying hydrolysable tannins, and Almond (Prunus dulcis (Mill.) D.A. 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APPENDIX

Table A1–Mean and range of polyphenols quantitated in whole almond (mg/100 g)

Class Subclass Compound Mean Min 25% Median 75% Max N Flavonoids Anthocyanin Cyanidin 2.20 0 0 2.20 4.40 4.40 2 Flavan-3-ol (+)-Catechin 5.40 0.1 0.3 1.51 5.72 36.6 32 Dihydrokaempferol 2.74 0.04 0.06 0.15 6.71 9.8 12 Dihydroquercetin 1.02 0.51 0.53 1.00 1.53 1.60 6 (-)-Epicatechin 2.11 0.03 0.33 0.72 1.93 26.6 42 Epicatechin gallate 2.60 2.60 2.60 2.60 2.60 2.60 1 Epicatechin glycoside 0 Gallocatechin gallate 0.39 0.31 0.31 0.39 0.46 0.46 2 Sum ﬂavan-3-ols 16.5 3.59 4.13 8.57 23.4 82.1 97 Flavanone Eriodictyol 0.41 0.30 0.33 0.44 0.46 0.46 4 Eridictyol-7-O-glucoside 0 Naringenin 2.19 0.02 0.10 0.13 3.44 9.74 17 Naringenin-7-O-glucoside 0.77 0.07 0.11 0.16 0.23 5.88 14 Sum ﬂavanones 3.37 0.39 0.54 0.73 4.13 16.1 35 Flavonol Isorhamnetin 0.62 0.09 0.10 0.12 1.32 3.20 14 Isorhamnetin-3-O-galactoside 0.59 0.30 0.45 0.58 0.71 0.92 8 Isorhamnetin-3-O-glucoside 9.27 0.11 1.60 11.8 14.9 14.9 6

r 366 ComprehensiveReviewsinFoodScienceandFoodSafety Vol.16,2017 C 2017 Institute of Food Technologists® Almond polyphenols . . .

Table A1–Mean and range of polyphenols quantitated in whole almond (mg/100 g)

Class Subclass Compound Mean Min 25% Median 75% Max N Isorhamnetin-3-O-rutinoside 22.9 1.12 4.64 14.7 36.0 74.1 16 Kaempferol 0.11 0.00 0.00 0.02 0.18 0.49 22 Kaempferol-3-O-galactoside 0.30 0.00 0.00 0.01 0.21 2.17 10 Kaempferol-3-O-glucoside 0.58 0.01 0.01 0.02 0.20 3.77 12 Kaempferol-3-O-rutinoside 5.18 0.13 0.60 0.99 10.1 23.3 16 Morin 0 Quercetin 0.29 0.00 0.02 0.03 0.14 3.58 18 Quercetin-3-O-galactoside 0.91 0.24 0.65 0.94 1.25 1.37 12 Quercetin-3-O-glucoside 0.09 0.04 0.05 0.08 0.14 0.16 10 Quercetin-3-O-rutinoside 0.62 0.02 0.22 0.40 1.09 1.66 18 Sum ﬂavonols 41.5 2.06 8.34 29.7 66.2 130 162 Phenolic Hydroxybenzoic p-Hydroxybenzoic acid 0.15 0.00 0.00 0.01 0.12 1.23 12 acids/aldehydes acid 5-Hydroxybenzoic acid 0 gallic acid 0.39 0.34 0.34 0.39 0.44 0.45 4 Protocatechuic acid 0.90 0.13 0.21 0.27 0.45 6.19 11 Vanillic acid 0.17 0.10 0.12 0.15 0.24 0.30 8 Sum 1.61 0.57 0.67 0.82 1.25 8.17 35 Hydroxybenzoic p-Hydroxybenzaldehyde 0 aldehyde Protocatehcuic aldehyde 3.78 2.52 2.68 3.41 5.24 5.77 4 Vanillin 0 Sum 3.78 2.52 2.68 3.41 5.24 5.77 4 Hydroxycinnamic Chlorogenic acid 0.96 0.00 0.00 0.60 2.29 2.29 3 acid 5,5-Diferulate 0 8,5-Diferulate (benzofuran) 0 Caffeic acid 0.13 0.11 0.11 0.13 0.15 0.15 4 o-Coumaric acid 0.48 0.22 0.26 0.50 0.67 0.69 4 p-Coumaric acid 0.28 0.01 0.05 0.32 0.51 0.59 7 Ferulic acid 1.80 1.28 1.37 1.87 2.14 2.15 4 Sinapic acid 0 Sum 3.65 1.62 1.79 3.42 5.76 5.87 22 Proanthocyanidins A-type dimers 0 A-type trimers 0 Procyanidin B1 4.49 1.69 1.69 4.49 7.28 7.28 2 Procyanidin B2 2.57 0.03 0.80 1.20 3.67 8.30 11 Procyanidin B3 0.32 0.19 0.19 0.32 0.45 0.45 2 Procyanidin B3 + B1 0 Procyanidin B5 0.22 0.00 0.00 0.22 0.43 0.43 2 Procyanidin B7 0.85 0.28 0.28 0.85 1.43 1.43 2 Procyanidin C1 0 Sum 8.45 2.19 2.96 7.08 13.26 17.89 19 Tannins Ellagitannins 54.7 53.3 53.3 53.4 57.4 57.4 3 Gallotannins 27.4 19.6 19.6 28.5 34.1 34.1 3 PAC dimers 11.4 4.00 4.00 11.35 18.7 18.7 2 PAC trimers 8.35 2.70 8.35 14.0 14.0 14.0 2 PAC 4–6mers 29.2 7.00 7.00 29.2 51.4 51.4 2 PAC 7–10mers 30.8 9.60 9.60 30.8 52.0 52.0 2 PAC polymers 82.4 43.9 43.9 82.4 121 121 2 Sum 244 140 146 250 349 349 16 Minor (μg) Bioachanin A 25.0 25.0 25.0 25.0 25.0 25.0 1 Daidzein 2.10 2.10 2.10 2.10 2.10 2.10 1 Coumesterol 1.50 1.50 1.50 1.50 1.50 1.50 1 Formonenetin 0.80 0.80 0.80 0.80 0.80 0.80 1 Genistein 7.50 1.00 1.00 7.50 14.0 14.0 2 Glycitein 0.60 0.60 0.60 0.60 0.60 0.60 1 Polydatin 7.72 7.19 7.19 7.44 8.52 8.52 3 Piceatannol + oxyresveratrol 1.72 0.90 0.90 1.71 2.56 2.55 3 (−)-7-Hydroxymatairesinol 27.0 27.0 27.0 27.0 27.0 27.0 1 7-Hydroxysecoisolariciresinol 10.0 10.0 10.0 10.0 10.0 10.0 1 Cyclolariciresinol 103 103 103 103 103 103 1 (+)-Lariciresinol 131 32.0 32.0 131 230 230 2 (−)-Matairesinol 12.2 0.30 0.30 12.2 24.0 24.0 2 (+)-pinoresinol 110 9.00 9.00 110 210 210 2 (+)-sesamin 82.0 82.0 82.0 82.0 82.0 82.0 1 (−)-Socoisolariciresinol 159 159 159 159 159 159 1 (+)-Syringaresinol 40.0 40.0 40.0 40.0 40.0 40.0 1 Sum 721 501 501 721 940 940 25

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Table A2–Polyphenol content and range reported in almond skins (mg/100 g)

Class Subclass Compound Flavonoids anthocyanin cyanidin Mean Min 25% Median 75% Max N Flavan-3-ol (+)-Catechin 6.33 0.69 2.01 4.11 9.46 18.4 30 Dihydrokaempferol 5.68 4.99 4.99 6.02 6.02 6.02 3 Dihydroquercetin 0.45 0.00 0.00 0.00 0.90 1.61 11 (−)-Epicatechin 3.60 0.13 0.82 2.50 6.10 11.0 29 Epicatechin gallate 0 Epicatechin glycoside 0 Gallocatechin gallate 0 Sum 16.1 5.81 7.82 12.6 22.5 37.0 73 Flavanone Eriodictyol 0.28 0.00 0.20 0.25 0.38 0.78 20 Eridictyol-7-O-glucoside 0.38 0.04 0.09 0.17 0.26 3.38 17 Naringenin 2.56 0.03 0.39 0.82 1.34 20.6 25 Naringenin-7-O-glucoside 2.76 0.04 1.12 2.59 3.55 14.3 41 Sum 5.98 0.11 1.80 3.83 5.53 39.1 103 Flavonol Isorhamnetin 1.14 0.40 0.50 0.85 1.58 4.55 24 Isorhamnetin-3-O-galactoside 0 Isorhamnetin-3-O-glucoside 1.94 0.20 0.90 1.39 1.56 16.9 27 Isorhamnetin-3-O-rutinoside 28.7 0.53 5.30 14.1 55.6 75.7 31 Kaempferol 0.41 0.01 0.20 0.30 0.42 1.25 25 Kaempferol-3-O-galactoside 1.00 0.72 0.72 1.14 1.15 1.15 3 Kaempferol-3-O-glucoside 3.31 0.00 0.0 0.62 2.12 39.0 26 kaempferol-3-O-rutinoside 6.94 0.10 1.28 3.18 9.53 23.9 35 Morin 0 Quercetin 0.31 0.03 0.15 0.31 0.44 0.70 25 Quercetin-3-O-galactoside 0.37 0.00 0.00 0.00 0.99 1.34 11 Quercetin-3-O-glucoside 0.22 0.00 0.10 0.17 0.28 0.90 21 Quercetin-3-O-rutinoside 5.00 0.00 0.00 0.45 2.08 41.2 21 Sum 49.3 1.99 9.15 22.5 75.8 207 249 Phenolic acids/aldehydes Hydroxybenzoic acid p-Hydroxybenzoic acid 0.63 0.03 0.31 0.53 0.72 1.90 23 5-Hydroxybenzoic acid 3.65 0.90 3.65 6.40 6.40 6.40 2 Gallic acid 0.67 0.00 0.06 0.53 1.41 1.61 4 Protocatechuic acid 1.99 0.04 0.88 1.72 3.20 4.46 26 Vanillic acid 1.37 0.01 0.76 1.16 1.85 5.81 22 Sum 8.31 0.98 5.66 10.3 13.6 20.2 77 Hydroxybenzoic aldehyde p-Hydroxybenzaldehyde 0 Protocatehcuic aldehyde 1.19 0.25 0.65 1.16 1.69 2.17 13 Vanillin Sum 1.19 0.25 0.65 1.16 1.69 2.17 13 Hydroxycinnamic acid Chlorogenic acid 0.92 0.17 0.20 0.38 0.88 9.57 21 5,5-Diferulate 0 8,5-Diferulate (benzofuran) 0 Caffeic acid 1.17 0.00 0.07 0.73 2.70 3.21 4 o-Coumaric acid 0 p-Coumaric acid 0.07 0.00 0.00 0.07 0.09 0.37 15 Ferulic acid 0 Sinapic acid 0 Sum 2.16 0.17 0.27 1.18 3.67 13.2 40 Proanthocyanidins A-type dimers 1.46 0.67 1.01 1.28 2.06 2.55 11 A-type trimers 0.32 0.16 0.19 0.30 0.43 0.53 11 Procyanidin B1 0 Procyanidin B2 1.15 0.23 0.53 0.96 1.61 3.39 13 Procyanidin B3 0 Procyanidin B3 + B1 1.68 0.30 1.27 1.59 2.10 2.96 14 Procyanidin B5 0.58 0.23 0.31 0.52 0.83 1.51 13 Procyanidin B7 0.98 0.37 0.48 0.56 1.42 2.47 13 Procyanidin C1 0.81 0.11 0.37 0.60 1.25 2.55 15 Sum 6.98 2.07 4.16 5.81 9.70 16.0 90

Table A3–USDA proanthocyanidin database value for almonds.a

Proanthocyanidin Mean (mg/100 g) Range (mg/100 g) N Dimers 9.2 4–18.7 17 Trimers 7.63 2.7–14 17 4–6mers 27.42 7–51.36 17 7–10mers 28.16 9.6–51.98 17 Polymers 80.26 43.86–120.94 8 aReferences: Gu and others (2003, 2004) and Xie and others (2012).

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