Introduction
============

Dietary guidance is consistent in recommending greater consumption of fruit and vegetables to promote health. Indeed, the 2015 Dietary Guidelines Advisory Committee report noted that greater fruit and vegetable intake was the only characteristic of dietary patterns that was consistently identified in their report in every conclusion statement across health outcomes ([@b1]). Although the report does not recommend specific types of fruit, there has been a growing body of evidence that the phytochemical composition of berry fruit may differentiate them from other fruits and underlie some of their putative benefits. Recent advances in analytical methods have improved the characterization of polyphenols in berry fruit and subsequently the data in food-composition and metabolomics databases that are essential for observational studies. Furthermore, the development of a standard reference material (SRM)[^14^](#fn4){ref-type="fn"} and matched placebos for use in clinical trials has provided an important and innovative component for the design and conduct of new randomized clinical trials. This review, prepared from the proceedings of the Cranberry Health Research Conference held in conjunction with the Berry Health Benefits Symposium in Madison, Wisconsin, 12--15 October 2015, focuses particularly on advances in the field during the last 5 y with regard to the gut microbiota and cardiometabolic health.

Cranberries and the Gut Microbiota
==================================

### Molecular mechanisms.

Much of the attention regarding the impact of cranberries on the gut microbiota has been directed to studies of the effect of cranberry extracts or juice on uropathogens and urinary tract infections (UTIs) ([@b2], [@b3]). However, this focus has expanded to encompass a broader range of the cranberry's antimicrobial, antifungal, and antiviral actions against *Helicobacter pylori* ([@b4]--[@b6]), *Streptococcus mutans* ([@b7]), *Porphyromonas gingivalis* ([@b8]), *Staphylococcus aureus* ([@b9]), *Pseudomonas aeruginosa* ([@b10]), *Cryptococcus neoformans* ([@b6]), *Haemophilus influenzae* ([@b11]), *Candida albicans* ([@b12], [@b13]), and extraintestinal pathogenic *Escherichia coli* (ExPEC) ([@b14]). Cranberry constituents, particularly the proanthocyanidins, flavonols, and hydroxycinnamic acids, may act against these pathogens by preventing bacterial adhesion and coaggregation, decreasing biofilm formation and/or reducing inflammation rather than via bactericidal activity. This expanding body of research includes in vitro, ex vivo, and animal studies that have suggested potential clinical effects and have helped to elucidate mechanisms of action as well as human studies that have shown physiologic effects ([@b3]--[@b5], [@b14]).

The antimicrobial properties of cranberry proanthocyanidins have been generally associated with their degree of polymerization (DP) and ratio of A- to B-type linkages. For example, by using an in vitro broth microdilution assay for growth inhibition of several yeast species, treatment of cultures with cranberry fractions of varying composition showed that cranberry proanthocyanidin fractions with a larger DP were found to be more effective than those with a smaller DP at inhibiting the growth of *Candida* spp. ([@b12]). In comparing primarily A-type proanthocyanidins from cranberries with primarily B-type proanthocyanidins from apples, Feliciano et al. ([@b15]) found that, although both increased agglutination and reduced epithelial cell invasion by ExPEC, the strongest effects were associated with a higher percentage of A-type linkages. This observation is consistent with other research that showed that A-type proanthocyanidins interact most strongly with bacterial virulence factors and more effectively decrease bacterial motility ([@b16], [@b17]).

### Microbiota biofilm.

The prevention of biofilm formation, an early step in the development of infection, through interference in the coaggregation of bacteria is a well-documented antimicrobial mechanism of cranberry proanthocyanidins. The extensively hydroxylated structure of proanthocyanidins encourages intermolecular hydrogen bonding, allowing smaller molecules to aggregate and interact with receptors on cell surfaces. Thus, many studies of high-molecular-weight nondialyzable material from cranberry juice concentrate reveal potent antiadhesion activity with microbial species, including those found in the oral cavity, stomach, small intestine, and colon ([@b6], [@b11], [@b14], [@b18], [@b19]). However, although purified cranberry proanthocyanidins are more effective in some antimicrobial assays than are crude or mixed extracts, several studies suggest that other compounds in cranberry possess antibacterial properties that alone or in combination with proanthocyanidins may enhance overall protection against infection. For example, Pinzón-Arango et al. ([@b20]) exposed *E. coli* to cranberry juice cocktail (CJC) or cranberry proanthocyanidins over 48 h and found that the proanthocyanidins reduced whereas the CJC completely eliminated biofilm formation. Candidate CJC constituents may include nonphenolic compounds such as isoprenoids like ursolic acid and xyloglucans, hemicellulose oligosaccharides found in high-molecular-weight nondialyzable fractions ([@b21]). Hotchkiss et al. ([@b22]) found that arabinoxyloglucans isolated from pectinase-treated cranberry hulls prevented the adhesion of *E. coli* strains to bladder and colonic epithelial cells in vitro.

Bacterial adhesion to cells and other surfaces involves basic physical forces such as electrostatic and steric interactions, van der Waals forces, and surface charge, as well as both specific and nonspecific interactions of surface proteins and carbohydrates such as glucans, adhesins, and sugar-specific lectins ([@b23]--[@b25]). Using atomic force microscopy, Liu et al. ([@b26]) found that exposure to cranberry juice decreased the adhesion forces of P-fimbriated *E. coli* (HB101pDC1) and altered the conformation and length of the P-fimbriae. Pinzón-Arango et al. ([@b24]) found that these fimbrial changes were reversible, even for cultures grown in the presence of cranberry juice. de Llano et al. ([@b27]) showed the efficacy of colonic metabolites of cranberry polyphenols, including hydroxylated benzoic and phenylacetic acids, in inhibiting the adhesion and biofilm formation of uropathogenic *E. coli* to bladder epithelial cells, a relation that underscores the critical need to elucidate the role of the gut microbiota in transforming cranberry polyphenols to bioactive and bioavailable compounds.

### Gut microbiota metabolism and function.

The gut microbiota is now appreciated as a critical factor in nutrition and health, influencing the bioavailability and metabolism of food components and affecting body systems, including brain and immune functions. The integrity of the gut mucosal barrier is essential for maintaining a chemical and physical barrier against food, environmental antigens, and microbes ([@b28], [@b29]). Goblet cells migrate up the villi after differentiating from crypt stem cells and turn over with the epithelial layer every 3--5 d. Goblet cells secrete mucins, particularly mucin 2 (Muc-2), that contribute substantially to the maintenance of mucosal integrity ([@b30]). Mucin secretion is regulated by a complex network of cholinergic stimulation and T-helper 2 (Th2) cytokines IL-4 and IL-13 ([@b31]--[@b35]).

Dysfunction of the gut barrier and dysbiosis have been associated with typical Western diets high in saturated fat and low in fiber and phytochemicals, patterns that may lead to increased permeability of bacterial LPS and a pathogen-associated molecular pattern that stimulates innate immune responses in macrophages, neutrophils, endothelial cells, and adipocytes. LPS plays a role in acute infection-related inflammatory responses and is found in blood and tissues with both postprandial and chronic inflammation ([@b36]--[@b39]). With the use of mice (CEABAC10) that express human carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), Martinez-Medina et al. ([@b37]) found that a high-fat, high-sugar diet increased intestinal permeability and TNF-α secretion, which resulted in a greater ability of adherent-invasive *E. coli* (AIEC) to colonize gut mucosa and induce inflammation. This diet also induced gut barrier dysfunction reflected by reduced levels of Muc-2 mRNA, increased permeability of 4-kDa fluorescein isothiocyanate-dextran, and decreased numbers of goblet cells. It is worth noting that AIEC may contribute substantially to the etiology of Crohn disease, an inflammatory bowel disease in which CEACAM6 is overexpressed on the apical surface of ileum epithelium ([@b40]). Furthermore, variant AIEC type 1 pili adhere to CEACAM6, a key step in the colonization of the ileum and chronic inflammation present in Crohn disease. In addition, after feeding mice a high-fat, high-sugar diet, Anhê et al. ([@b41]) reported that the addition of a cranberry extract attenuated the consequent chronic inflammation associated with gut barrier dysfunction, including reductions in plasma LPS, cyclooxygenase-2, and TNF-α. Furthermore, the ratio of NF-κB to inhibitor κB was significantly lower in the jejunal tissue of the mice fed cranberry extract relative to the mice fed the high-fat, high-sugar diet. Also suggesting the capacity of cranberry polyphenols to reduce intestinal oxidative stress and inflammation, in vitro experiments with Caco-2/15 intestinal cells by Denis et al. ([@b42]) revealed positive but differential effects of low-, medium-, and high-molecular-mass polyphenols from cranberries on oxidative stress, proinflammatory cytokines, NF-κB activation, and nuclear factor E2-related factor 2 (Nrf2) downregulation, as well as PPAR-γ coactivator 1α.

Interestingly, the effects of high-fat, high-sugar diets on gut barrier function in mice are similar to those observed in animal models of parenteral nutrition and elemental enteral nutrition (EEN) ([@b43], [@b44]). EEN induces dysfunction of gut-associated lymphoid tissue, including decreased lymphocytes in Peyer's patch and reduced tissue Th2 cytokines, and suppresses mucosal barrier function when compared with normal nutrition ([@b43], [@b45]--[@b48]). The addition of cranberry proanthocyanidins to EEN was found to increase ileal tissue IL-4 and IL-13 concentrations, goblet cell number and size, and the secretion of intestinal Muc-2, attenuating the impairment of the mucosal barrier integrity after EEN alone ([@b44]). Pierre et al. ([@b43]) reported that the addition of cranberry proanthocyanidins significantly supported other indexes of gut-associated lymphoid tissue function impaired by EEN in mice, indicated in part by decreased Peyer's patch lymphocytes and lower concentrations of tissue Th2 cytokines. Cranberry proanthocyanidins also helped to restore the EEN-induced decreases in polymeric Ig receptor, a transport protein involved in enterocyte transcytosis of secretory IgA (sIgA) from B cells in the lamina propria into the intestinal lumen. EEN decreases in luminal concentrations of sIgA were attenuated by cranberry proanthocyanidins; intestinal sIgA opsonizes bacterial antigens such as the virulence factors of pathogenic *E. coli*, rendering them less viable and more susceptible to killing by lymphocytes. The addition of cranberry proanthocyanidins also significantly prevented EEN-induced decreases in tissue IL-4 and phosphorylated signal transducers and activators of transcription 6 (STAT6).

Clinical studies are necessary to determine whether the results from these mouse models can be translated to the capacity of cranberry phytochemicals to reduce diet-induced intestinal inflammation in humans. Interestingly, there is limited evidence suggesting an effect of cranberry on systemic immune function in humans, which may be partly mediated via gut metabolism of cranberry polyphenols. For example, a randomized, double-blind placebo-controlled study documented increased ex vivo proliferation of γδ-T cells, immune cells located within the epithelium of the gastrointestinal and reproductive tracts, after the consumption of a cranberry beverage for 10 wk ([@b49]).

### ExPEC in the gut.

Although ExPEC generally do not cause acute enteric disease, their colonization in the gut increases the risk of subsequent extraintestinal infection, including UTIs, septicemia, surgical wound infections, and neonatal meningitis ([@b50], [@b51]). ExPEC attach to and invade epithelial cells through adhesins expressed on type I pili (protein FimH) and P fimbriae (fimbrial adhesin PapG) and persist inside the host cell in vacuoles where they may evade immune detection. ExPEC, uropathogenic *E. coli*, and the AIEC associated with Crohn disease have similar virulence factors and are within the same *E. coli* phylogroups (B2 and D) ([@b40]). These phylogroups differ from enteropathogenic *E. coli* and Shiga toxin--producing *E. coli*, such as O157:H7, because enteropathogenic *E. coli* and Shiga toxin--producing *E. coli* cause acute intestinal disease and produce attaching and effacing lesions of the intestinal epithelium. Gut colonization by ExPEC is a likely cause of a chronic inflammatory state because ExPEC may evade immune detection and colonize enterocytes. The continuous presence of *E. coli* LPS in the gut mucosa may cause chronic intestinal inflammation. Although ExPEC have a meaningful impact on public health via their consequences on morbidity and mortality, they have not received concordant attention because they have been highly susceptible to antibiotics. However, 20--45% of ExPEC have become resistant to first-line antibiotics such as cephalosporins, fluoroquinolones, and trimethoprim-sulfamethoxazole ([@b52], [@b53]). Thus, it is becoming critical to appreciate and further investigate the potential role for dietary bioactive components in reducing such infections.

Recently, Feliciano et al. ([@b15]) showed that A-type proanthocyanidins have greater bioactivity than B-type proanthocyanidins for increasing ExPEC agglutination and decreasing their invasion (and subsequent colonization) of gut epithelial cells, an important observation for the elucidation of the effect of cranberry proanthocyanidins on UTIs. As suggested by Feliciano et al. ([@b15]) and other studies described above, decreasing intestinal colonization and associated inflammation may be achieved by usual serving sizes of cranberry juice without the requirement for absorption of its constituent proanthocyanidins into the circulation or their appearance in the urine. It is important to note that recent randomized clinical trials have confirmed and extended the body of evidence showing cranberry's bacterial antiadhesion activity in urine ex vivo ([@b3], [@b54]), its capacity to reduce the recurrence of UTIs ([@b55]), and its therapeutic efficacy in preventing UTIs in gynecologic surgery patients after catheter removal ([@b56]). Nonetheless, additional research that uses similarly relevant ex vivo and in vivo models can be used to substantiate the structure-function relation of A-type proanthocyanidins to intestinal and extraintestinal infections and to develop preventive and therapeutic strategies against increasingly antibiotic-resistant classes of pathogens ([@b57], [@b58]). Such an effort could be advanced by the availability of a cranberry SRM as discussed below.

Cranberries and Cardiometabolic Health
======================================

A limited but growing number of clinical research studies ([@b59]--[@b72]) have focused on cardiometabolic health ([**Tables 1**](#tbl1){ref-type="table"} and [**2**](#tbl2){ref-type="table"}). The most commonly examined risk factors for cardiometabolic conditions in these studies have included serum lipid profiles, blood pressure (BP), endothelial function, glucoregulation, and a variety of biomarkers of inflammation and oxidative stress. Although the results of this research have generally been promising, a clear and consistent picture of this emerging area is confounded by sometimes marked differences in the cranberry products (cranberry juices, dried cranberries, and cranberry extracts) and doses used, as well as the characteristics of the study populations ([@b2], [@b73]). Although few animal model studies have examined this topic, Kim et al. ([@b74]--[@b76]) reported that 5% cranberry powder added to atherogenic diets with or without intraperitoneal LPS administration produced positive effects on serum lipids, proinflammatory cytokines, oxidative stress, and antioxidant capacity in rodents.

###### 

Summary of randomized placebo-controlled trials on the cardiometabolic effects of cranberry[^1^](#tblfn1){ref-type="table-fn"}

  Study, year (ref)                 Intervention        Population                       Age,[^2^](#tblfn2){ref-type="table-fn"} y   *n*   Study design   Duration, wk   Dose/d       Polyphenol content                     Outcomes
  --------------------------------- ------------------- -------------------------------- ------------------------------------------- ----- -------------- -------------- ------------ -------------------------------------- -----------------------------------------------------------------------------------------------------------------------------------------
  Lee et al., 2008 ([@b59])         Cranberry extract   Adults with T2D                  65 ± 1                                      30    Parallel       12             500 mg × 3   Not reported                           ↓: TC, LDL-C; NC: FBG, HbA1c, SBP, DBP, TGs, HDL-C, oxLDL-C, insulin, HOMA-IR, CRP
  Shidfar et al., 2012 ([@b60])     CJ                  Adults with T2D                  55 ± 9                                      58    Parallel       12             240 mL       Not reported                           ↓: FBG, apoB; ↑: paraoxonase-1 activity; NC: apo A-1, Lp(a)
  Novotny et al., 2015 ([@b61])     LC CJ               Healthy adults                   50 ± 11                                     56    Parallel       8              480 mL       346 mg TPs, 21 mg ACNs, 235 mg PACs    ↓: DBP, FBG, HOMA-IR, TGs, CRP; NC: SBP, insulin, HOMA-β, TC, LDL-C, HDL-C, apo A-1, apo A-2, apoB, ICAM, VCAM
  Basu et al., 2011 ([@b62])        LC CJ, 27%          Adults with metabolic syndrome   52 ± 8                                      31    Parallel       8              480 mL       458 mg TPs, 25 mg ACNs                 ↓: oxLDL-C, malondialdehyde, 4-hydroxynonenal; NC: SBP, DPB, FBG, TC, LDL-C, HDL-C, VLDL-C, TGs, IL-6, CRP, plasma antioxidant capacity
  Dohadwala et al., 2011 ([@b63])   CJ, 54%             Adults with CAD                  63 ± 9                                      44    Crossover      4              480 mL       835 mg TPs, 94 mg ACNs                 ↓: HDL-C, carotid femoral PWV; NC: SBP, DPB, FBG, insulin, HOMA-IR, TC, LDL-C, TGs, carotid radial PWV, CRP, ICAM-1
  Flammer et al., 2013 ([@b64])     LC CJ, 54%          Adults with CVD risk factors     49 ± 16                                     69    Parallel       16             460 mL       800 mg TPs, 69 mg ACNs, 1224 mg PACs   NC: SBP, DBP, TC, HDL-C, TGs, AIX, pulse pressure, heart rate, reactive hyperemia index, CRP, ICAM, VCAM, IL-6, TNF-α, oxLDL-C
  Ruel et al., 2013 ([@b65])        LC CJ, 27%          Healthy overweight men           45 ± 10                                     35    Crossover      4              500 mL       400 mg TPs, 21 mg ACNs                 NC: SBP, DBP, mean arterial pressure, heart rate, AIX, global endothelial function, NOx, uric acid, oxLDL-C, ICAM-1, VCAM-1, E-selectin

ACN, anthocyanin; AIX, augmentation index; CAD, coronary artery disease; CJ, cranberry juice; CRP, C-reactive protein; CVD, cardiovascular disease; DBP, diastolic blood pressure; FBG, fasting blood glucose; HbA1c, glycated hemoglobin; HDL-C, HDL cholesterol; HOMA-β, homeostatic model assessment of β cell function; ICAM, intercellular adhesion molecule; Lp(a), lipoprotein a; LC, low-calorie; LDL-C, LDL cholesterol; NC, no significant change; NOx, nitrate/nitrite; oxLDL-C, oxidized LDL cholesterol; PAC, proanthocyanidin; PWV, pulse-wave velocity; ref, reference; SBP, systolic blood pressure; TC, total cholesterol; TP, total polyphenol; T2D, type 2 diabetes; VCAM, vascular cell adhesion molecule; VLDL-C, VLDL cholesterol; ↓, significantly different decrease from placebo group; ↑, significantly different increase from placebo group.

Values are means ± SDs.

###### 

Summary of open-label trials on the effects of cranberry on cardiometabolic markers[^1^](#tblfn3){ref-type="table-fn"}

  Study, year (ref)              Intervention          Population                       Age, y                                                                *n*   Study design                                Duration                   Dose/d                Polyphenol content                         Outcomes
  ------------------------------ --------------------- -------------------------------- --------------------------------------------------------------------- ----- ------------------------------------------- -------------------------- --------------------- ------------------------------------------ --------------------------------------------------------------------------------------------------------------
  Ruel et al., 2006 ([@b66])     LC CJ, 27%            Healthy sedentary men            51 ± 10[^2^](#tblfn4){ref-type="table-fn"}                            30    Crossover                                   4 wk/dose                  125, 250, 500 mL      100 mg TPs, 5 mg ACNs, 74 mg PACs/125 mL   ↓: TC:HDL-C, antioxidant capacity, NOx; ↑: HDL-C; NC: TC, LDL-C, TGs, apo A-I, apoB
  Ruel et al., 2008 ([@b67])     LC CJ, 27%            Healthy sedentary men            51 ± 10                                                               30    Crossover                                   4 wk/dose                  125, 250, 500 mL      100 mg TPs, 5 mg ACNs, 74 mg PACs/125 mL   ↓: SBP, HDL-C, oxLDL-C, ICAM-1, VCAM-1; NC: DBP, heart rate, TC, LDL-C, TGs, apoB, E-selectin
  Ruel et al., 2009 ([@b68])     LC CJ, 27%            Healthy sedentary men            51 ± 10                                                               30    Crossover                                   4 wk/dose                  125, 250, 500 mL      100 mg TPs, 5 mg ACNs, 74 mg PACs/125 mL   ↓: SBP, MMP-9, NOx; NC: DBP, mean arterial BP
  Ruel et al., 2005 ([@b69])     LC CJ, 27%            Healthy men                      38 ± 8                                                                21    Single-arm intervention; no control group   14 d                       7 mL/kg body weight   Not reported                               ↓: oxLDL-C; ↑: antioxidant capacity; NC:SBP, DBP, TC, LDL-C, HDL-C, TC:HDL-C, TGs, apoB, LDL-C particle size
  Wilson et al., 2008 ([@b70])   CJ, 27%; LC CJ, 27%   Adults with T2D and obesity      65 ± 2                                                                12    Crossover                                   Single dose                Not reported          Not reported for dosage given              CJ vs. LC CJ---↑: glucose at 30 and 60 min, insulin at 60 min; NC: glucose, insulin at 120 min
  Wilson et al., 2010 ([@b71])   SDC, LC SDC, RC, WB   Adults with T2D                  62 ± 2                                                                13    Crossover                                   30, 60, and 120 min/dose   40 g each             Phenolic and PAC profiles reported         WB, SDC, LC SDC vs. RC---↑: glucose at 30, 60, 120 min; insulin at 30, 60 min; glucose AUC; insulin AUC
  Simão et al., 2013 ([@b72])    LC CJ                 Adults with metabolic syndrome   48.5 (control), 51.0 (cranberry)[^3^](#tblfn5){ref-type="table-fn"}   56    Parallel; no-intervention control           60 d                       700 mL                364 mg TPs, 231 mg PACs                    ↑: Adiponectin; ↓: lipoperoxidation, protein oxidation; NC: CRP, IL-1, IL-6, TNF-α, folic acid, homocysteine

ACN, anthocyanin; BP, blood pressure; CJ, cranberry juice; CRP, C-reactive protein; DBP, diastolic blood pressure; HDL-C, HDL cholesterol; ICAM, intercellular adhesion molecule; LC, low-calorie; LDL-C, LDL cholesterol; MMP, metalloproteinase; NC, no significant change; NOx, nitrites/nitrates; oxLDL-C, oxidized LDL cholesterol; PAC, proanthocyanidin; RC, raw cranberries; ref, reference; SBP, systolic blood pressure; SDC, sweetened dried cranberries; TC, total cholesterol; TP, total polyphenol; T2D, type 2 diabetes; VCAM, vascular cell adhesion molecule; WB, white bread; ↓, significantly different decrease from baseline, between groups, and/or across increasing doses; ↑, significantly different increase from baseline, between groups, and/or across increasing doses.

Mean ± SD (all such values).

Median ages in control and treatment groups.

### Lipid profile.

Early reports by Ruel et al. ([@b66]--[@b68]) found that interventions with low-calorie cranberry juice were associated with increases in plasma HDL cholesterol as well as with reductions in plasma oxidized LDL cholesterol, adhesion molecules, and matrix metalloproteinase 9. Lee et al. ([@b59]) showed a reduction in both LDL cholesterol and total cholesterol in a trial in 30 patients with type 2 diabetes (T2D) who consumed cranberry extract supplements daily for 12 wk. In a double-blind, placebo-controlled trial, Shidfar et al. ([@b60]) reported that 58 men with T2D who consumed 1 cup cranberry juice/d for 12 wk experienced decreases in apoB and increases in apo A-1 and paraoxonase-1, although data on LDL, HDL, and total cholesterol were not reported. In an 8-wk randomized clinical trial of low-calorie cranberry juice consumption by 56 healthy adults, Novotny et al. ([@b61]) found that TGs were significantly decreased in the cranberry group whereas other elements of the lipid profile were unchanged.

### BP.

Previous studies of the effect of cranberry juice on BP suggested a potential benefit on BP ([@b67], [@b69]). More recent studies also examined changes in BP after cranberry intake ([@b59], [@b61]--[@b65]). The durations of these studies ranged from 1 to 4 mo and tested intakes of total polyphenols ranging from 346 to 835 mg/d; and study populations were heterogeneous, including subjects with obesity, metabolic syndrome, T2D, coronary artery disease (CAD), and risk factors for cardiovascular disease (CVD), as well as healthy volunteers. With daily doses of CJC increasing every 4 wk from 0--125 to 250--500 mL, systolic BP decreased by 3 mm Hg with the 500-mL intervention compared with baseline in obese men ([@b67]). Of the more recent studies, only the study performed in healthy individuals and with the lowest dose of polyphenols showed an improvement in BP, with a reduction of 4.7 mm Hg in diastolic BP achieved after 8 wk of daily supplementation ([@b61]).

### Endothelial function.

Endothelial dysfunction, often characterized by a decrease in nitric oxide production and impaired flow-mediated vasodilation (FMD), is a critical factor underlying the development and progression of atherosclerosis ([@b77]). In a randomized controlled trial with a crossover design, Dohadwala et al. ([@b63]) found that daily supplementation with cranberry juice for 4 wk did not improve FMD or peripheral artery tonometry in 44 patients with CAD, although an uncontrolled pilot study in a subset of the same population showed a modest improvement in FMD 4 h after an acute dose of cranberry juice. In a 4-wk trial with a cranberry juice drink, Flammer et al. ([@b64]) found no significant changes in peripheral artery tonometry in individuals with endothelial dysfunction and other CVD risk factors. Further research on the effect of cranberries on measures of vascular reactivity is required in healthy individuals examining both the dose-response and time course of the intervention.

Recently, in a clinical study of 10 healthy adults, Feliciano et al. ([@b78]) identified and quantified by ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry analysis a total of 60 cranberry-derived phenolic metabolites in plasma and urine after the acute ingestion of cranberry juice containing 787 mg polyphenols. These metabolites included sulfates of pyrogallol, valerolactone, benzoic acids, phenylacetic acids, and glucuronides of flavonols, as well as sulfates and glucuronides of cinnamic acids. Their concentrations ranged from in the low nanomolars to the high micromolars depending on the compound. Among these 60 phenolic metabolites, 12 were found to be independent predictors of time- (0--6 h) dependent increases in FMD after an acute dose (range: 409--1909 mg total polyphenols) ([@b79]). These results indicate that cranberry polyphenols can acutely increase endothelial function in healthy individuals. Arterial stiffness, commonly assessed by pulse-wave velocity or the augmentation index (a measure of the enhancement of central aortic pressure by a reflected pulse wave), is an established risk factor for CVD ([@b80]--[@b82]). In their randomized clinical trial of patients with CAD, Dohadwala et al. ([@b63]) found that a 4-wk intervention with cranberry juice significantly reduced the carotid-femoral pulse-wave velocity. However, no changes in the augmentation index were observed by Ruel et al. ([@b65]) after 4 wk of supplementation with cranberry juice in 35 volunteers presenting with obesity and other cardiovascular risk factors.

### Glucoregulation.

Berry fruit polyphenols have been shown by in vitro experiments and animal models to inhibit carbohydrate digestion and glucose absorption in the intestine, stimulate insulin secretion from β cells in the pancreas, regulate glucose release from the liver, and activate insulin receptors and glucose uptake in insulin-sensitive tissues ([@b83]--[@b85]). Emerging clinical evidence suggests that dietary modification to increase polyphenol intakes from whole-food sources can lead to improved glycemic control in T2D ([@b86], [@b87]). While exploring the antidiabetic effects of a cranberry extract in high-fat, high-sugar--fed mice, Anhê et al. ([@b41]) found a decrease in glucose-induced hyperinsulinemia and improved insulin sensitivity along with a reduction in weight gain and visceral obesity. As noted above, along with these improvements, the cranberry extract also altered the gut microbiome by increasing mucin-degrading bacteria. In light of the evolving link between the gut microbiota and diabetes, these findings provide an important connection between the studies documenting the effects of cranberry on gut barrier function and the potential to reverse the dysbiosis and metabolic inflammation underlying diabetes ([@b88]).

Human studies testing low-calorie cranberry juice and unsweetened dried cranberries were shown to produce favorable acute postprandial glycemic responses in adults with T2D ([@b70], [@b71]). However, the limited number of longer-term studies in patients with T2D generated discordant outcomes. Shidfar et al. ([@b60]) reported that daily cranberry juice consumed for 12 wk by 58 male patients with T2D induced significant decreases in fasting blood glucose when compared with the placebo group. In contrast, in a trial of 30 patients with T2D, Lee et al. ([@b59]) found no impact of daily supplementation with cranberry extracts for 12 wk on fasting blood glucose or glycated hemoglobin. In a diet therapy intervention in 27 adults with T2D, Chambers and Camire ([@b89]) found no significant effect of treatment with cranberry extract on measures of glycemia. However, in 12 healthy volunteers in a randomized crossover trial, Törrönen et al. ([@b90]) found that a berry puree containing cranberries was able to delay the postprandial plasma response to sucrose. Clinical trials in patients with metabolic syndrome have suggested some benefit associated with cranberry intervention but not specifically on outcomes of glycemic control ([@b62], [@b72], [@b91]).

### Biomarkers of inflammation.

In their analysis of observational data collected from NHANES, Duffey and Sutherland ([@b92], [@b93]) found inverse associations of popular polyphenol-containing beverages, such as cranberry juice, with obesity and inflammation. In their 8-wk randomized clinical trial, Novotny et al. ([@b61]) showed a reduction in C-reactive protein (CRP) after daily consumption of cranberry juice. In a clinical trial of 56 subjects with metabolic syndrome, Simão et al. ([@b72]) reported that daily intake of low-calorie cranberry juice for 8 wk had no significant effect on proinflammatory cytokines IL-1, IL-6, and TNF-α but did reduce biomarkers of lipid peroxidation and advanced oxidation protein products. Similarly, in an 8-wk study of 36 patients with metabolic syndrome, Basu et al. ([@b62]) observed increases in plasma biomarkers of antioxidant capacity and decreases in lipid peroxidation after the daily consumption of low-calorie cranberry juice. These results are consistent with in vitro experiments showing that cranberry polyphenols decreased the generation of reactive oxygen species and lipid peroxides and increased glutathione peroxidase activity and phospho-c-Jun N-terminal kinase ([@b94]).

Cranberries and Health: Knowledge Gaps
======================================

The recent growing body of research on cranberries and health is a part of the emerging evidence from in vitro, animal model, and human studies of plant polyphenols as protective dietary agents that act both directly and indirectly via their metabolites and/or interactions with the gut microbiota. Improvements in these research approaches, particularly in analytical methods as diverse as MS and gene-sequencing methods for microbial communities, are making important contributions to our understanding of polyphenol mechanisms and functions.

However, an important need in cranberry health research is the consistent use of a fully characterized SRM to help promote the generation of more readily comparable and replicable research protocols. SRMs have been available for some other polyphenol-rich foods, including highbush blueberries ([@b95]) and California table grapes ([@b96]), for in vitro, animal, and human studies. Although SRMs for cranberries have been developed by the National Institute of Standards and Technology and the Office of Dietary Supplements of the NIH, these materials are intended for the validation of analytical methods and quality assurance for in-house control materials. Furthermore, these SRMs are not accompanied by matching placebos for use in research studies. Wide availability of cranberry SRMs in sufficient quantities to conduct in vivo human health research remained lacking until recently. Because concerns for study accuracy and quality were raised because of the diversity of cranberry products available commercially and in research protocols ([@b97]), The Cranberry Institute undertook the development of a cranberry reference material in 2014 to ensure the authenticity and consistency of cranberry products used in research on human health.

The first question to be answered was whether the SRM would be developed from whole fruit or one of the many processed forms in which cranberry is consumed. The impact of processing, particularly juicing, on the phytochemical content and profile of fresh cranberries has been characterized ([@b98], [@b99]). Grace et al. ([@b100]) compared fresh and freeze-dried cranberries to cranberry-containing commercial products including juices (from concentrate and not from concentrate), sweetened dried cranberries, and cranberry sauces (homemade and commercially canned). Cranberry skins and flesh were cross-compared for anthocyanin and proanthocyanidin content. Proanthocyanidins were typically higher in skins than in flesh with the exception of the proanthocyanidin A-2 dimer. Anthocyanin and proanthocyanidin concentrations were lower in juice reconstituted from concentrate. In general, the retention of proanthocyanidins in processed cranberries was found to be robust, whereas anthocyanins were sensitive to degradation. Grace et al. ([@b101]) explored ways to better concentrate and stabilize cranberry bioactive compounds via complexing concentrated juices with proteins isolated from soy, hemp, peanuts, and peas for formulating both beverage and solid-food products. By using an in vitro model to simulate digestion, Ribnicky et al. ([@b102]) were able to show that protein complexes with blueberry polyphenols remained more intact and bioaccessible than the free bioactive compounds.

To retain and encourage the study of the complete phytochemical profile of cranberry, the SRM is a freeze-dried whole-cranberry powder (FWCP). It is produced from a blend of cranberry varieties grown in Wisconsin and approximating the proportion available in the marketplace (i.e., 56% Stevens plus 11% each of Ben Lear, Grygleski, Pilgrim, and HyRed varieties for the first batch produced in 2015). The berries are individually frozen after harvest, freeze-dried, and ground into powder form. Silicon dioxide (3% total volume of powder) is added as an anticaking agent. The production process is fully documented from harvest to storage. Each 50 g (0.5 cup) of whole cranberries produces ∼4.5 g FWCP.

Complete specifications for each nutrient and phytochemical ingredient were prepared by using a series of assays, including matrix-assisted laser desorption/ionization time-of-flight MS for authentication of proanthocyanidins ([@b103], [@b104]), 4-(dimethylamino)cinnamaldehyde assay for quantification of soluble proanthocyanidins ([@b57], [@b103], [@b105]), and butanol-hydrochloric acid for quantification of insoluble proanthocyanidins as well as characterization of efficacy via an established in vitro antiadhesion assay and microbiological testing.

Accurate quantification of proanthocyanidins for health research is essential but also problematic because proanthocyanidins are complex polydispersed hetero-oligomers ([@b57]). Previously, the procyanidin A2 (ProA2) dimer was recommended as the standard of choice for proanthocyanidin analysis in the 4-(dimethylamino)cinnamaldehyde assay because cranberry proanthocyanidins contain ≥1 "A-type" interflavan bonds ([@b106]). However, current evidence shows that the use of the ProA2 dimer as a standard for quantification of complex proanthocyanidin oligomers results in a serious underestimation of proanthocyanidins ([@b107]). To address this problem, a cranberry proanthocyanidin standard (c-PAC), reflective of the structural heterogeneity of proanthocyanidins found in fresh cranberry (i.e., DP, hydroxylation pattern, and ratio of A- to B-type interflavan bonds), was developed. The use of the c-PAC to quantify proanthocyanidin content in FWCP resulted in values that were 3.6 times those determined by ProA2. Thus, adoption of this c-PAC standard reflects an improvement over the use of ProA2 for the accurate quantification of cranberry proanthocyanidins ([@b105]). Because these findings were only recently published, the soluble proanthocyanidin content of the FWCP is reported as both c-PAC and ProA2 equivalents, allowing researchers time to adopt the new methodology. The c-PAC was also used to quantify the FWCP insoluble proanthocyanidins by the butanol-hydrochloric acid method.

The polyphenol content of the FWCP includes the following: 28.35 mg total polyphenols (gallic acid equivalents)/g, 31.20 mg total soluble proanthocyanidins (c-PACs)/g, 8.77 mg soluble proanthocyanidins (ProA2)/g, 10.38 mg insoluble proanthocyanidins (c-PACs)/g, 5.98 mg anthocyanins (cyanidin-3-galactoside equivalents)/g, 9.01 mg flavonols (quercetin-3-rhamnoside equivalents)/g, and 1.81-mg hydroxycinnamic acids (caffeic acid equivalents)/g ([@b108]). The FWCP processing and packaging facilities are compliant with FDA regulations. A suitable placebo was created from a blend of maltodextrin, citric acid, artificial flavoring, fructose, and food-grade coloring agents ([@b109]). Calcium silicate is added to the FWCP and placebo as a flow agent.

The use of the FWCP should help overcome some of the critical limitations associated with past studies that used uncharacterized or only partly characterized cranberry foods or extracts. Recipes for the administration of FWCP and placebo in human studies have been developed and are made freely available to researchers. Like other studies of whole foods, it is recommended that protocols that use the FWCP not apply this material directly to target tissues, with some possible exceptions such as oral and gastrointestinal cells. In vitro and ex vivo research approaches should consider the use of metabolite(s) on the basis of their likely bioavailability to these tissues, an approach not often followed in early studies of polyphenol-rich foods and extracts. The design of clinical trials that use the FWCP should also be informed by human bioavailability data generated from studies of other cranberry foods and extracts ([**Table 3**](#tbl3){ref-type="table"}) ([@b110]--[@b114]), although some consideration should be directed to results from animal models ([@b115]). However, because the product matrices and pharmacokinetic characteristics of these other products will undoubtedly differ, new studies on the absorption, metabolism, and elimination of the bioactive compounds in the FWCP must be undertaken. Although the availability of the FWCP as an SRM for clinical research may help ensure the consistency and full characterization of the cranberry intervention, the need to perform reasonable dose-response and time-course studies for each health-related outcome remains an important priority, as does the need to develop biomarkers of compliance to the intervention.

###### 

Summary of human trials investigating the bioavailability and pharmacokinetic variables of cranberry[^1^](#tblfn6){ref-type="table-fn"}

  Study, year (ref)                 Population                      *n*   Cranberry product and dose                                 Compounds studied                                  Variables studied                                                   Timing of measurements
  --------------------------------- ------------------------------- ----- ---------------------------------------------------------- -------------------------------------------------- ------------------------------------------------------------------- --------------------------------------------------------------------
  Feliciano et al., 2016 ([@b78])   Healthy young men               10    450 mL CJ                                                  Phenolic metabolites                               Plasma AUC, Cmax, Tmax, % urinary recovery                          Plasma: 1, 2, 4, 6, 8, and 24 h; urine: 0--8 h, 8--24 h
  Zhang and Zuo, 2004 ([@b110])     Healthy adults                  1     1800 mL, 27% CJ                                            Flavonoids, phenolic acids, benzoic acids          Plasma and urine concentrations                                     0, 45, and 270 min
  Milbury et al., 2010 ([@b111])    Adults aged 62 ± 8 y with CAD   15    480 mL 54% CJ (835 mg TPs, 94.47 mg ACNs)                  ACNs                                               Plasma AUC, Cmax, Tmax, T1/2 % urinary recovery                     0--4 h
  Iswaldi et al., 2013 ([@b112])    Adults aged 25--40 y            4     0.6 mL/kg cranberry syrup                                  Polyphenols, phase I and II phenolic metabolites   Urine concentration                                                 Urine: 0, 2, 4, and 6 h
  McKay et al., 2015 ([@b113])      Adults aged ≥50 y               10    54% CJ                                                     Flavonoids, phenolic acids, PACs                   Plasma AUC, Cmax, Tmax, antioxidant capacity, urine concentration   Plasma: 0.25, 0.5, 1--6, and 10 h; urine: 12, 14, 16, 18, and 20 h
  Walsh et al., 2016 ([@b114])      Healthy women aged 20--30 y     5     237 mL cranberry beverage (140 mg PACs); weekly for 7 wk   PACs                                               Urine concentration                                                 24 h

ACN, anthocyanin, CAD, coronary artery disease; CJ, cranberry juice; Cmax, maximal plasma concentration; PAC, proanthocyanidin; ref, reference; T1/2, biological half-life; Tmax, time to maximal plasma concentration.

Summary
=======

Cranberry juice, dried cranberries, and various cranberry extracts have been shown via in vitro, animal model, and human studies to possess an array of biochemical and physiologic activities mediated by their phytochemical constituents. Although the greatest research focus has been reasonably placed on their rich content of polyphenols, emerging evidence of their actions on the gut microbiota and cardiometabolic functions suggests that attention is also warranted on their synergy with cranberry phenolic acids, isoprenoids, and oligosaccharides. Acting in high concentrations within the gastrointestinal lumen, these cranberry compounds may act to quench reactive oxygen species, modulate inflammatory pathways, adhere to carbohydrates and proteins on bacterial surfaces, exert prebiotic effects, and alter the dynamic cross-talk between intestinal epithelial cells and the gut microbiota. These actions may underlie not only the antimicrobial effects of cranberries but their role in the complex pathogenesis of UTIs and inflammatory bowel diseases. The importance of these relations beyond the gastrointestinal tract has grown substantially with the recognition of the broad role that the gut microbiota plays in regulating energy homeostasis, glucose and lipid metabolism, and systemic inflammation, all factors associated with the maintenance of cardiometabolic health.

Further substantiating the actions and mechanisms of cranberry constituents can best be accomplished by taking advantage of recent advances in cranberry research. For example, efforts to identify biomarkers of compliance to clinical protocols, as well as their relation to physiologic and health outcomes, may evolve from improved understanding of cranberry constituents (e.g., the specific nature of proanthocyanidin interflavan bonds and DP, as well as a more robust phytochemical profile) and the numerous bioactive catabolites arising from the biotransformation of cranberry constituents by the gut microbiota and phase I, II, and III metabolism pathways. Furthermore, a greater degree of accuracy, consistency, and quality of new studies has become possible with the availability of a fully characterized FWCP and matched placebo as SRMs.

JBB and CDT outlined and coedited this article in addition to their contribution to the writing; JBB, AB, CGK, MAL, CCN, JAN, JDR, AR-M, and CDT contributed to the writing and review of the manuscript. All authors read and approved the final manuscript. We appreciate the excellent moderation of the Cranberry Health Research Conference presentations and panel discussions by Amy Howell (Rutgers University), Christina Khoo (Ocean Spray Cranberries, Inc), and the active participation by the panelists: C-Y Oliver Chen (Tufts University), André Marette (Université Laval), Yeonwha Park (University of Massachusetts at Amherst), Navindra Seeram (University of Rhode Island), David Sela (University of Massachusetts at Amherst), and Christine Wu (University of Illinois at Chicago).

Abbreviations used: AIEC, adherent-invasive *Escherichia coli*; BP, blood pressure; CAD, coronary artery disease; CEACAM, carcinoembryonic antigen-related cell adhesion molecule; CJC, cranberry juice cocktail; c-PAC, cranberry proanthocyanidin standard; CRP, C-reactive protein; CVD, cardiovascular disease; DP, degree of polymerization; EEN, elemental enteral nutrition; ExPEC, extraintestinal pathogenic *Escherichia coli*; FimH, protein FimH; FMD, flow-mediated vasodilation; FWCP, freeze-dried whole-cranberry powder; Muc-2, mucin 2; Nrf2, nuclear factor E2-related factor 2; PapG, fimbrial adhesin PapG; ProA2, procyanidin A2; sIgA, secretory IgA; SRM, standard reference material; STAT6, signal transducers and activators of transcription 6; Th2, T-helper 2; T2D, type 2 diabetes; UTI, urinary tract infection.

[^1]: Published in a supplement to *Advances in Nutrition*. Presented at the Cranberry Health Research Conference, held in Madison, Wisconsin, 12 October 2015 and sponsored by the Cranberry Institute (CI), the US Cranberry Marketing Committee (CMC), and the American Cranberry Growers Association. The Supplement Coordinator for this supplement was Cheryl D Toner. Supplement Coordinator disclosure: Cheryl D Toner is the contracted Health Research Coordinator for the CI and a consultant to the CI and the CMC. Publication costs for this supplement were defrayed in part by the payment of page charges. This publication must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact. The opinions expressed in this publication are those of the author(s) and are not attributable to the sponsors or the publisher, Editor, or Editorial Board of *Advances in Nutrition*.

[^2]: Author disclosures: The Cranberry Institute (CI) provided travel expense reimbursement to all authors and provided an honorarium to each author except for JA Novotny and CD Toner. CD Toner is a consultant to the CI and the CMC and formerly to the Juice Products Association. JB Blumberg is a member of the Scientific Advisory Board of the CI and the CMC and has received research support from the CI and Ocean Spray Cranberries, Inc. CG Krueger, JD Reed, and A Rodriguez-Mateos have received research support from the CI. JA Novotny has received research support from Ocean Spray Cranberries, Inc.

[^3]: This is a free access article, distributed under terms (<http://www.nutrition.org/publications/guidelines-and-policies/license/>) that permit unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
