Health benefits of walnut polyphenols: An exploration beyond their lipid profile

ABSTRACT Walnuts are commonly found in our diet and have been recognized for their nutritious properties for a long time. Traditionally, walnuts have been known for their lipid profile, which has been linked to a wide array of biological properties and health-promoting effects. In addition to essential fatty acids, walnuts contain a variety of other bioactive compounds, such as vitamin E and polyphenols. Among common foods and beverages, walnuts represent one of the most important sources of polyphenols, hence their effect over human health warrants attention. The main polyphenol in walnuts is pedunculagin, an ellagitannin. After consumption, ellagitannins are hydrolyzed to release ellagic acid, which is converted by gut microflora to urolithin A and other derivatives such as urolithins B, C, and D. Ellagitannins possess well known antioxidant and anti-inflammatory bioactivity, and several studies have assessed the potential role of ellagitannins against disease initiation and progression, including cancer, cardiovascular, and neurodegenerative diseases. The purpose of this review is to summarize current available information relating to the potential effect of walnut polyphenols in health maintenance and disease prevention.


Introduction
Walnuts (Juglans regia L.) have been consumed for a long time as a highly nutritious food in many parts of the world and are an important component of the Mediterranean diet (Bull o, Lamuela-Ravent os, & Salas-Salvad o, 2011). Recently, these have gathered attention for their health-promoting properties, which have been reported to improve lifestylerelated diseases. The health benefits of walnuts are attributed to several of their nutrients, such as v-3 fatty acids (Ros et al., 2004), vitamin E (Maguire, O'Sullivan, Galvin, O'Connor, & O'Brien, 2004), and dietary fiber. In addition to these nutrients, walnuts are rich in plant sterols and especially in polyphenols, as it has been reported recently (Vinson and Cai, 2012;Regueiro et al., 2014). The implications of polyphenols in human health are numerous and include beneficial effects over several diseased states, including cardiovascular system dysfunction and damage (Estruch et al., 2013), metabolic syndrome (Murase et al., 2011), diabetes (Li et al., 2011), various inflammation-related pathologies (Konstantinidou et al., 2010), and cancer (Thangapazham et al., 2007;Nkondjock, 2009). In addition, polyphenols have been recently described as potentially beneficial compounds for neuroprotection (Granzotto and Zatta, 2014), and against aging (Granzotto and Zatta, 2014;Peng et al., 2014). Ellagitannins, the polyphenols typically found in walnuts, and their derived metabolites possess a wide range of biological activities, which suggest that they could have beneficial effects on human health (Esp ın et al., 2013). The purpose of this review is to summarize the available information about the health-promoting effects of walnut polyphenols, mainly pedunculagin and its metabolites, ellagic acid, and urolithins.

Chemistry and metabolism of walnut polyphenols
It has been reported by Vinson and Cai (2012) that walnuts represent the seventh largest source of total polyphenols among common foods and beverages based on their serving size. A handful of walnuts, about 50 g, has significantly more total polyphenols than a glass of apple juice (240 mL), a milk chocolate bar (43 g), or a glass of red wine (150 mL), which are all common food sources of polyphenols (Anderson et al., 2001). Moreover, walnut's total polyphenols were significantly higher compared to other nuts such as almonds, hazelnuts, pistachios, and peanuts (Abe, Lajolo, & Genovese, 2012;Vinson and Cai, 2012). Indeed, the total reported range of polyphenol contents is from 1576 to 2499 mg per 100 g of walnuts (Vinson and Cai, 2012). In addition, walnut polyphenol extracts also exhibited high antioxidant potential, with an antioxidant capacity of 21.4 § 2.0 mmol TE/100 g and 25.7 § 2.1 mmol TE/10 g measured by ABTSC and DPPH assays, respectively (Regueiro et al., 2014).
It is well documented that the most abundant polyphenols in walnuts are ellagitannins, mainly pedunculagin ( Fig. 1) (Cerd a et al., 2005;Regueiro et al., 2014). Ellagitannins exhibit structural diversity according to food source. Although regardless of food source, ellagitannins are characterized by one or more hexahydroxydiphenoyl moieties esterified to a polyol (Regueiro et al., 2014). Previous studies of rat intestinal contents showed that ellagitannins could be hydrolyzed to ellagic acid at a pH found in the small intestine and cecum (Daniel et al., 1989;Garcia-Muñoz and Vaillant, 2014). The presence of free ellagic acid in human plasma could be due to its release from the hydrolysis of ellagitannins, enabled by physiological pH and/or gut microbiota. Ellagic acid is further metabolized by gut flora to form urolithins, mainly urolithin A and B (Landete, 2011), which are probably synthesized in the colon (Garcia-Muñoz and Vaillant, 2014) (Fig. 1). These urolithins circulate in blood and can reach many of the target organs where the effects of ellagitannins are observed (Larrosa et al., 2006b). The occurrence of ellagitannins and ellagic acid in the bloodstream is almost negligible, but their derived metabolites, urolithins, can reach a concentration at micromolar levels in plasma (Larrosa et al., 2006a). It is important to note that urolithin concentration in plasma and urine after ingestion of ellagitannin-rich foods can differ between individuals due to individual differences in gut microbiota (Garcia-Muñoz and Vaillant, 2014). In addition to urolithin concentration, specific urolithin production can vary between individuals. A recent study identified three urolithin-producing phenotypes related to the type of urolithins produced after consumption of ellagitannin food sources (Tom as-Barber an et al., 2014).

Effects on oxidative stress
Oxidative stress can be defined as an imbalance between free radical and reactive metabolites such as reactive oxygen species (ROS), production, and their elimination. This lack of balance can lead to cell damage, potentially creating an impact in an organism as a whole ( Dura ckov a, 2010). Inflammation can be caused because of oxidative stress, and although excess of ROS is not the only cause, inflammation resulting from oxidative stress is the origin of many diseases (Martinon, 2010). Typical examples are dyslipidemia (Hopps et al., 2010), thrombosis (Leopold and Loscalzo, 2009), metabolic syndrome (Hopps et al., 2010), type 2 diabetes (Kaneto et al., 2010),nonalcoholic steatohepatitis (NASH) (Hijona et al., 2010), macular degeneration (Augustin and Kirchhof, 2009), neurodegenerative diseases such as Alzheimer's (Candore et al., 2010), and cancer (Hussain et al., 2003). During inflammation, mast cells and leukocytes are recruited to the site of damage, which leads to a "respiratory burst" due to an increased uptake of oxygen, and thus an increased release and accumulation of ROS (Hussain et al., 2003). On the other hand, inflammatory cells also produce soluble mediators, such as metabolites of arachidonic acid, cytokines, and chemokines, which act by further recruiting inflammatory cells to the site of damage and producing species that are more reactive. Over the years, epidemiologic and observational evidence has encouraged belief in the use of bioactive compounds with antioxidant potential for disease prevention (Stanner et al., 2007). Considering the previously mentioned antioxidant capacity of ellagitannins, it is interesting to explore the potential of these compounds in disease prevention.
Ultraviolet (UV) radiation from the sun is a potent environmental risk factor in the pathogenesis of skin damage, much of the damage caused by ultraviolet-A (UVA) irradiation is associated with oxidative stress. Ellagic acid may be useful for the treatment of UV-induced skin damage. In an in vitro study, Hseu et al (2012) showed that an ellagic acid pre-treatment markedly increased HaCaT human keratocyte cell viability and suppressed UVA-induced ROS generation and malondialdehyde (MDA) formation. Moreover, ellagic acid pre-treatment prevented UVA-induced DNA damage and significantly inhibited the UVA-induced apoptosis of HaCaT cells. The antioxidant potential of ellagic acid was directly correlated with the increased expression of heme oxygenase 1 (HO-1) and superoxide dismutase (SOD), which was followed by the downregulation of kelch-like ECH-associated protein 1 (Keap1) and the augmented nuclear translocation and transcriptional activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) with or without UVA irradiation (Hseu et al., 2012). These results support the role of ellagic acid in activating a response against oxidative stress (Fig. 2).
Ellagic acid has also shown a protective role against nicotine-induced toxicity in rat peripheral blood lymphocytes (Sudheer et al., 2007). Lymphocytes incubated with nicotine showed a significant increase in the levels of lipid peroxidation index, severity in DNA damage, and micronuclei number. These effects were modulated by ellagic acid treatment. Antioxidant status was also significantly depleted in nicotine-treated group, which was effectively restored by ellagic acid incubation (Sudheer et al., 2007).

Anti-inflammatory effects
The protective role of ellagitannins and its metabolites against acute inflammation has been explored in in vivo and in vitro models (Umesalma and Sudhandiran, 2010;Ahad et al., 2014;El-Shitany et al., 2014) (Fig. 3). Ellagic acid exhibited a potent anti-inflammatory effect against carrageenan-induced inflammation (El-Shitany et al., 2014). The mechanisms by which ellagic acid is protected against inflammation could be linked to the reduction of inflammatory molecules such as nitric oxide (NO), MDA, interleukin-1 beta (IL-1b), tumor necrosis factor alpha (TNF-a), cyclo-oxygenase 2(COX-2), and nuclear factor-kB (NF-kB) expression, and the induction of glutathione (GSH) and IL-10 production (El-Shitany et al., 2014). Urolithins have also been linked to cause a protective effect in acute inflammation. Ishimoto and colleagues (2011) investigated the anti-inflammatory role of urolithin A in carrageenan-induced paw edema in mice. The volume of paw edema was reduced 1 h after the oral administration of urolithin A. In addition, plasma exhibited significant oxygen radical antioxidant capacity (ORAC) scores in treated mice, correlating with high plasma levels of the unconjugated form 1 h after the oral administration of urolithin A (Ishimoto et al., 2011). These studies show that both ellagic acid and its bioactive metabolite urolithin A exert anti-inflammatory effects by several mechanisms, including reduction of oxidation and inflammatory cytokines.
Of particular interest is the role of ellagic acid and their metabolites on colon inflammation, which can help elucidate the potential role of ellagitannin-containing foods, such as walnuts, on preventing gut inflammatory diseases. Colon fibroblasts CCD18-Co cells were exposed to a mixture of urolithins A and B and ellagic acid at concentrations comparable with those found in the colon (Gim enez-Bastida et al., 2012). The effects on fibroblast migration and monocyte adhesion were also determined. The mixture of polyphenol metabolites significantly inhibited colon fibroblast migration by about 70% and monocyte adhesion to fibroblasts by about 50%. These effects were parallel with a significant down-regulation Figure 2. Scheme of the role of ellagitannins on antioxidant-related processes in the cell. HO-1: heme oxygenase 1, Keap1: kelch-like ECH-associated protein 1, Maf: musculoaponeurotic fibrosarcoma oncogene, Nrf2: nuclear factor (erythroid-derived 2)-like 2, SOD: superoxide dismutase.
of inflammatory markers, such as prostaglandin E2 (PGE2), plasminogen activator inhibitor 1 (PAI-1), and IL-1b, as well as other key regulators of cell migration and adhesion (Gim enez-Bastida et al., 2012). The results show that a combination of ellagitannin metabolites at concentrations achievable in the intestines after the consumption of ellagitannin-containing foods, such as pomegranate or walnuts, was able to moderately improve the inflammatory response of colon fibroblasts, and suggest that consumption of ellagitannin-containing foods has potential beneficial effects on gut inflammatory diseases. In addition, a recent study further confirms the protective effect that urolithins have over intestinal inflammation using RAW 264.7 murine macrophages as a model (Piwowarski et al., 2015). Urolithins A, B, and C decreased NO production via inhibition of iNOS protein and expression of IL-1b, TNF-a, and IL-6 (Piwowarski et al., 2015).
Inflammatory processes are associated with several pathological conditions. Available data indicate that the development of diabetic nephropathy, a serious complication confronted by diabetic patients, is also linked to inflammation. A study performed in type 2 diabetic Wistar albino rats evaluated the nephro-protective effects of ellagic acid (Ahad et al., 2014). Ellagic acid treatment for 16 weeks after induction of diabetes significantly attenuated renal dysfunction and oxidative stress. It also significantly inhibited renal NF-kB activation, lowered renal pathology, suppressed transforming growth factor-beta (TGF-b), and fibronectin expressions in renal tissues. Moreover, ellagic acid significantly reduced the serum levels of proinflammatory cytokines, IL-1b, IL-6, and TNF-a (Ahad et al., 2014). Thus, these authors concluded that ellagic acid exerted a renal protective effect in type 2 diabetic rats by a multifactorial approach through anti-hyperglycemic, anti-glycative, antioxidant, and anti-inflammatory effects. Considering this evidence, the anti-inflammatory properties of ellagitannins potentially exert a protective effect against a wide range of pathologies and their complications.

Estrogenic and androgenic interactions
There has been increased interest in studying the activity of phytoestrogens due to their potential health benefits. The modulation of hormone receptors by phenolic dietary components has been widely studied. Many polyphenols, including isoflavanones, flavanones, and stilbenes, have shown phytoestrogenic activity (Mantena et al., 2006;Thangapazham et al., 2007;Tu et al., 2011;Wang et al., 2011). Dietary polyphenols may also be the precursors of bioactive compounds with estrogenic activity and ellagitannins, particularly their metabolites, urolithins A and B, can be included in this group (Fig. 4). A study has assessed the potential activity of urolithins as hormone-disruptive molecules, exerting both estrogenic and anti-estrogenic activity (Larrosa et al., 2006a). This work performed structure analyses, which revealed that urolithins A and B exhibited structural characteristics that made these molecules able to bind with the aand b-estrogen receptors (ERs). An estrogen receptor competitive binding assay in MCF-7 breast cancer cells showed that both urolithins had affinity for ERa and ERb receptors, although urolithin A bound more effectively than urolithin B, and had higher affinity for ERa than for ERb receptor. Urolithins showed weaker estrogenic activity than other phytoestrogens, such as daidzein, genistein, and enterolactone, but they both displayed slightly higher anti-estrogenic activity than the previously mentioned phytoestrogens, dose-dependently antagonizing the growth-promotion effect of 17-b-estradiol. In addition, our group also explored the hormone-related activity of urolithins A and B using LNCaP androgen-dependent prostate adenocarcinoma cells. Considering the reported anti-androgenic activity of other phytoestrogens, such as isoflavanones, and the phytoestrogenic effect of urolithins observed by Larrosa and colleagues (2006a), we hypothesized that urolithins A and B could potentially interact with the androgen receptor (AR). We observed a significant decrease in AR mRNA and protein levels after treatment with urolithins at several time points. Electrophoretic mobility shift assays also revealed that urolithins decreased AR binding to androgen response elements, which in turn resulted in a decreased expression of AR-regulated genes such as klk3, which encodes for prostate-specific antigen (PSA) (Fig. 4) (S anchez-Gonz alez et al., 2014). Considering the aforementioned evidence, it is possible to conclude that urolithins can play a key role in the modulation of hormone and hormonereceptor-dependent diseases such as breast and prostate cancers.

Health benefits of walnut polyphenols and their derived metabolites
Cancer Polyphenols exert their anticancer effects by several mechanisms, such as the reduction of pro-oxidative effect of carcinogenic agents (Duthie & Dobson, 1999;Owen et al., 2000), modulation of cancer cell signaling and cell cycle progression (Corona et al., 2007;Corona et al., 2009;Khan and Mukhtar, 2013), promotion of apoptosis (Fabiani et al., 2002;Mantena et al., 2006), and modulation of enzymatic activities (Adams et al., 2010). Polyphenols have also been shown to act on multiple targets in pathways not only related to cellular proliferation and death (Fini et al., 2008) but also in inflammation (Kang et al., 2011), angiogenesis (Granci et al., 2010), and drug and radiation resistance (Garg et al., 2005). In particular, the effect that walnut polyphenols have on cancer prevention has been studied widely, showing promising results (Spaccarotella et al., 2008;Reiter et al., 2013;Hardman, 2014;S anchez-Gonz alez et al., 2015). Both in vitro and in vivo studies that assess the role of ellagitannins in several molecular pathways related to cancer initiation, development, and progression have been performed.

Prostate
Prostate cancer is the second most frequently diagnosed cancer and the sixth leading cause of cancer deaths among men worldwide (American Cancer Society, 2011). An important target in prostate cancer is the androgen receptor, which is required for the development and progression of prostate carcinogenesis. As previously mentioned, our research group has assessed the effect that urolithins have on the modulation of androgen receptor, which is fundamental in prostate cancer progression. We were able to determine inhibition in both AR and PSA gene expression and a decrease in protein levels after incubating androgen-dependent LNCaP prostate adenocarcinoma cells with urolithins A and B. In addition, we were able to determine that urolithins inhibited the binding of AR to androgen response elements, which are transcription factors necessary for the transcription of genes such as PSA. An induction of apoptosis and a decrease in the anti-apoptotic protein BCL-2 were also observed (S anchez-Gonz alez et al., 2014). In addition, after further study using a functional genomics approach, we also determined CDKN1A, which encodes for p21 protein, as a node gene of urolithin A activity in a prostate cancer cell model. Upon validation, a significant increase in anti-proliferative p21 mRNA and protein levels was observed. In addition, an increased activity of apoptotic enzymes, caspases 3 and 7, was seen (S anchez-Gonz alez et al., 2015). Other authors have also observed anti-apoptotic effects in other prostate cancer cells such as PC3 and DU145, which are androgen-independent, demonstrating that walnut polyphenols negatively affect prostate cancer cell viability via different mechanisms (Vicinanza et al., 2013).
Another established target in prostate cancer chemoprevention is a cytochrome P450 enzyme, CYP1B1. Compounds inhibiting CYP1B1 activity are contemplated to exert beneficial effects at three stages of prostate cancer development, that is, initiation, progression, and development of drug resistance. Urolithins, especially urolithin A, were found to decrease CYP1B1 activity and protein expression in 22Rv1 prostate cancer cells (Kasimsetty et al., 2009). Furthermore, both walnut extracts and ellagitannins have also been linked to suppressing prostate cancer cell proliferation and inducing apoptosis (Losso et al., 2004;Alshatwi et al., 2012;Vicinanza et al., 2013;Naiki-Ito et al., 2015) in several models. These cell-based assays allow for the identification of molecular pathways where walnut polyphenols may exert their activity.
Animal and human studies on the potential link between walnuts and prostate cancer prevention are still limited, but the results seem promising. Reiter and colleagues (2013) tested whether a walnut-enriched diet influenced the growth of prostate cancer xenografts growing in male nude mice. They found that the walnut-enriched diet reduced the number of tumors and the growth of LNCaP xenografts. These authors hypothesized that the most likely explanation for the finding that a walnut-enriched diet forestalled the growth of prostate tumors is that the inhibitory effect was a consequence of the combined actions of several phytochemicals, among them are the polyphenolic compounds. Similarly, another recent in vivo study found that prostate tumor weight and growth rate were reduced in the transgenic adenocarcinoma of mouse prostate (TRAMP) cancer model after treatment with a walnut diet (Davis et al., 2012). Like Reiter et al. (2013), the authors stated that the beneficial effects of a walnut-enriched diet probably represent the effects of multiple constituents in whole walnuts and not due to specific bioactive compounds such as fatty acids or tocopherols.
Human studies are still lacking, and a recent study points to the fact of a need to better design in vitro and in vivo approaches to mimic physiological conditions as closely as possible (Gonz alez-Sarr ıas et al., 2010a). This interventional study involving 63 patients with either benign prostate hyperplasia or prostate cancer divided subjects into three groups: controls, walnut intake (35-g walnuts/day), and pomegranate intake (200-mL pomegranate juice/day) for three days before prostate surgery. The main metabolite detected after consumption was urolithin A glucuronide. No apparent changes in the expression of CDKN1A, MKi-67, or c-Myc (all related to cancer cell proliferation) were found after consumption of either of the experimental food treatment. The results from this study demonstrated that conjugates of urolithins, specifically glucuronides, and dimethyl ellagic acid can reach and enter the human prostate gland upon consumption of ellagitannin-rich sources such as pomegranate juice and walnuts. Considering their results and the lack of changes in proliferation markers, these authors expressed the need to design better in vitro studies that should focus on the bioactivity and exposure time of the actual in vivo metabolites formed upon consumption of ellagitannins (Gonz alez-Sarr ıas et al., 2010a). Another clinical trial done in healthy men showed that consuming walnuts on a regular basis did not influence serum PSA levels, although it did improve biomarkers of prostate health (Spaccarotella et al., 2008); this indicates a positive effect of including walnuts as part of men's diet on prostate health.
As a conclusion, all the previously mentioned findings point out the need for studies using whole foods, such as walnuts, in human intervention trials to truly assess their effects on prostate cancer and to identify effective, food-based chemoprevention diets for prostate and other cancers.

Breast cancer
Considerable amount of research has focused on the role that food bioactive components have in the development and progression of breast cancer. In a study conducted by Hardman and colleagues (2011), female mice were fed with control or walnut-containing diets, and were followed for tumor development. Compared to a diet without walnuts, their consumption significantly reduced tumor incidence, number of tumors per mouse, and their size. Gene expression analyses indicated that a walnut diet altered expression of multiple genes associated with proliferation and differentiation of mammary epithelial cells. Although specific bioactive components were not tested, the results of this study indicate that walnut consumption could contribute to a healthy diet for the prevention of breast cancer.
It is well known that an important target in breast cancer research is the estrogen receptor, since estrogens have a critical role in the development and growth of breast cancer (Clemons and Goss, 2001). As such, research addressed to study the interaction of ellagitannins against estrogen positive breast cancer models is of interest. In an in vivo study, the role of ellagic acid over the expression of miRNAs related to breast cancer was evaluated using an ACI rat model, which develops mammary tumors upon estrogen E2 (estradiol) exposure. Recent reports have associated several miRNAs with estrogen receptors in breast cancers. Ellagic acid reversed the deregulation of a number or miRNAs, such as miR-375, miR-206, miR-182, miR-122, miR-127, and miR-183, detected after E2 incubation. It also modulated their target proteins, which include ERa, cyclin D1, RASD1, FoxO3a, FoxO1, cyclin G1, Bcl-w, and Bcl-2. These observations provide mechanistic insight into the molecular events behind the chemo-preventive action of ellagic acid, specifically in breast cancer (Munagala et al., 2013). Ellagic acid also exhibited the potential to down-regulate the 17b-estradiolinduced hTERT aCbC mRNA expression in MCF-7 estrogen sensitive breast carcinoma cells (Strati et al., 2009).
As previously mentioned, the estrogen receptor represents an important target for the treatment of hormone-dependent breast cancer with anti-estrogens due to the effect estrogen has on breast cancer cell growth and progression. The hormonelike activity reported for urolithins has been related to their interaction with the estrogen receptor in MCF-7 cells, showing a high binding affinity to this hormone receptor (Larrosa et al., 2006a), and exerting both estrogenic and anti-estrogenic effects. Therefore, urolithins can potentially antagonize the response of hormone-dependent breast cancer cells to estrogens. In addition, the potential anti-aromatase activity of ellagitanninderived compounds has been studied in MCF-7aro cells (ER-positive aromatase overexpressing cells) (Adams et al., 2010). The aromatase enzyme converts androgen to estrogen and as such plays a key role in breast carcinogenesis. They screened a panel of ellagitannin-derived compounds and identified six with anti-aromatase activity; Urolithin B was shown to most effectively inhibit enzymatic activity. Their results suggested that urolithin B likely inhibits the proliferation of MCF7aro breast cancer cells primarily through aromatase inhibition, and that anti-proliferative effects caused by the other test compounds may be caused by an aromatase-independent mechanism such as direct antagonism of estrogen receptor signaling or a combination of aromatase-dependent and independent mechanisms (Adams et al., 2010). Strong anti-proliferative activity was observed after ellagic acid incubation in MCF-7 and Hs 578T cell lines (Losso et al., 2004), although it is interesting to note that in this study cell lines from different cancer models were studied, and the most resistant were the breast cancer cells (Losso et al., 2004). Considering all the previously mentioned studies, the role of ellagitannins and derived metabolites in breast cancer prevention is promising. Additional studies could aid in further elucidating the molecular effects that ellagitannins have in breast cancer development and progression inhibition.

Colon cancer
Foods rich in ellagitannins could act against colon carcinogenesis as evidenced by several studies (Losso et al., 2004;Gonz alez-Sarr ıas et al., 2010b;Gim enez-Bastida et al., 2012;Qiu et al., 2013). Walnut leaves, green husks, and seed extracts showed concentration-dependent growth inhibition toward human kidney and colon cancer cells (Carvalho et al., 2010). Exposure of Caco-2 (colon adenocarcinoma) cells to ellagic acid and urolithins arrested cell growth at the S-and G2/M-phases, which could have been related to a decrease in the expression levels of MAPK signaling genes, tumor suppressors, and genes involved in cell cycle (Gonz alez-Sarr ıas et al., 2009). Another signaling pathway known to play a pivotal role in human colon carcinogenesis is the Wnt pathway. An inappropriate activation of this signaling cascade is observed in 90% of colorectal cancers. Wnt transcriptional activation was explored in a human 293T colon cancer cell line, cells were incubated with ellagitannin extracts from several food sources, including strawberries and pomegranate, and specific polyphenols, ellagic acid (63 mM) and urolithin A (39 mM). All extracts, ellagic acid, and urolithin A inhibited Wnt-dependent signaling, a promising effect against colon carcinogenesis (Sharma et al., 2010).
A walnut-containing diet has also been shown to inhibit colorectal cancer growth by suppressing angiogenesis. In an in vivo study using HT-29 cells (colon carcinoma) injected to mice, tumor growth rate was significantly slower in walnut-fed (27%) compared with corn oil-fed animals (Nagel et al., 2012). Consequently, final tumor weight was reduced by 33% versus control. Walnuts also reduced serum expression levels of angiogenesis factors, including vascular endothelial growth factor by 30%, and significantly decreased angiogenesis proved by CD34 staining. In addition to the anti-proliferative effects of walnut bioactive compounds, its main metabolite, urolithin A, has been shown to potentiate the effects of 5-Fluorouracil (5-FU, drug of first choice in colorectal cancer therapy) on colon cancer cells. This would suggest the need for lower 5-FU doses to achieve therapeutic effects, which could in turn reduce possible adverse effects of treatment and may indicate a role of ellagitannins as chemotherapy adjuvants (Gonz alez-Sarr ıas et al., 2015). Although, further studies are needed to confirm these findings in humans and explore underlying mechanisms in more detail, the previously mentioned studies highlight several targets modulated by ellagic acid and its metabolites in colon cancer.

Other cancers
Ellagitannins and their metabolites inhibit cancer cell growth, among other ways, through cell cycle arrest and stimulation of apoptosis. The anti-proliferative activities of ellagic acid and its metabolites have been evaluated in several human cancer cell lines, some of which have been previously mentioned (Adams et al., 2010;Vicinanza et al., 2013;S anchez-Gonz alez et al., 2014S anchez-Gonz alez et al., , 2015. Other studies have also evaluated the anti-proliferative activity of ellagitannins and other byproducts of their hydrolysis in a diverse range of cancer cell lines. These include an in vitro study conducted in a human bladder cancer cell line, which assessed anti-proliferative activity of four ellagitannin metabolites: urolithin A, urolithin B, 8-OMe-urolithin A, and ellagic acid. The results from this study suggested that these compounds could inhibit cell proliferation by p38-MAPK and/ or c-Jun-mediated caspase-3 activation and by the reduction of oxidative stress (Qiu et al., 2013). Other anti-cancer effects of ellagic acid have also been observed through PKC a gene down-regulation and decreased activity, resulting in a marked decrease in oxidative stress and cell viability in Dalton lymphoma bearing mice (Mishra and Vinayak, 2011), and in a study conducted in SH-SY5Y neuroblastoma cells ellagic acid incubation-induced anti-proliferative effects, including cell detachment, decreased cell viability, and induced apoptosis (Fjaeraa and Na nberg, 2009).

Cardiovascular disease
The characteristic lipid profile of walnuts has been suggested to reduce the risk of cardiovascular diseases, exerting its effect by decreasing total and LDL-cholesterol and increasing both HDL-cholesterol and the antioxidant defense system (Ros et al., 2004;Nergiz-€ Unal et al., 2013). However, recent studies have shown the potential cardio-protection effects of ellagitannins, which could be associated with the modulation of several parameters linked to cardiovascular health (Papoutsi et al., 2008;Spaccarotella et al., 2008;Larrosa et al., 2010). One of the strongest effects has been observed on oxidative stress and inflammation; in this sense, it is interesting to explore the potential role of ellagitannins over atherosclerosis.
The inflammatory process plays an important role in the pathogenesis of atherosclerosis through the interaction of endothelium with immune cells (Kaneto et al., 2010). Numerous signaling cascades have been elucidated and, among other functions, the inflammatory cytokine-induced adhesion molecules in the endothelium play a critical role in the inflammatory process and immune response (Han et al., 2007). Adhesion molecules, namely vascular cell adhesion molecule (VCAM)-1 and intracellular cell adhesion molecule (ICAM)-1, activated by inflammatory cytokines such as TNF-a, participate in the initiation of this interaction. Several lines of evidence support a crucial role of adhesion molecules in the development of atherosclerosis and plaque instability (Blankenberg et al., 2003). Papoutsi et al. (2008) examined the effect of a walnut extract and ellagic acid on VCAM-1 and ICAM-1 expression in human aortic endothelial cells. Cells were incubated with TNFa in the absence and presence of walnut extract or ellagic acid, both treatments significantly decreased TNF-a-induced endothelial expression of VCAM-1 and ICAM-1 (Papoutsi et al., 2008). Authors concluded that walnut polyphenols, mainly ellagic acid, show potential as anti-inflammatory substances and provided insight into the mechanism of how walnut intake may participate in cardio-protection by improving endothelial function.
Another important intervention of walnut polyphenols as modulators of cardiovascular disease is the protective effect they may have on the susceptibility of LDL to oxidative modification and ultimately on atherosclerosis. In a study conducted by Anderson et al. (2001), polyphenol-rich walnut extracts at expected physiologic concentration were studied and compared with ellagic acid for their ability to inhibit in vitro plasma and LDL oxidation during oxidative stress. LDL oxidation was significantly inhibited by both ellagic acid and walnut extract. These authors concluded that walnut polyphenols are effective inhibitors of in vitro plasma and LDL oxidation (Anderson et al., 2001), indicating that in addition to the favorable lipid profile of walnuts, their phenolic content must also be considered as a potential contributor to the apparent anti-atherogenic effect of walnuts (Casas-Agustench et al., 2011;Estruch et al., 2013).
Interestingly, ellagic acid has also been associated with a protective effect on myocardial infarction-induced damage. Wistar rats were used as a model to provide a scientific basis for the use of ellagic acid in preventing myocardial infarction (Mari Kannan and Darlin Quine, 2012). These authors also performed in vitro studies that confirmed the free radical scavenging and metal chelating activities of ellagic acid, which could be the mechanism responsible for protective action against mitochondrial damage in myocardial infarction.
The previously mentioned in vitro and in vivo studies provide the basis for elucidating the effect of walnut polyphenols over cardiovascular diseases. Human trials have also been performed, granting they do not directly assess the specific effect of polyphenols. Katz et al (2012) associated walnuts and their bioactive compounds to impact endothelial function favorably. In a randomized controlled crossover trial, using a population of overweight individuals with visceral adiposity, the effects of daily walnut consumption on endothelial function and other biomarkers of cardiac risk were investigated. Forty-six overweight adults, with elevated waist circumference and one or more additional signs of metabolic syndrome, were randomly assigned to two eight-week sequences of walnut-enriched ad libitum diet and ad libitum diet without walnuts, which were separated by a four-week washout period. The primary outcome measure was the change in flow-mediated vasodilation (FMD) of the brachial artery. In this study, FMD improved significantly from baseline when subjects consumed a walnutenriched diet as compared with the control diet. There was also a reduction in systolic blood pressure, and maintenance of the baseline anthropometric values was also observed. Although specific components of walnuts were not studied specifically, these authors concluded that the daily ingestion of 56 g of walnuts improves endothelial function in overweight adults with visceral adiposity (Katz et al., 2012). This study did not assess polyphenols in particular, but it is a good example of the positive cardiovascular health effects of walnuts as a diet component. It would be interesting to assess whether the individual bioactive compounds, of which walnuts are composed of, causes these effects, or whether it is a result of the interaction between the various phytochemicals they contain.

Neurodegenerative disease
Lifestyle factors greatly affect the progression of cognitive decline, with high-risk behaviors, including unhealthy diet, lack of exercise, smoking, and exposure to environmental toxins, leading to enhanced oxidative stress and inflammation. Although there is an urgent need to develop effective treatments for age-related cognitive decline and neurodegenerative disease, prevention strategies are underdeveloped. Thus, potential preventive effects of bioactive compounds commonly found in the diet should be explored. As mentioned previously in this review, polyphenolic compounds found in walnuts have both anti-inflammatory and anti-oxidant properties. Therefore, these compounds might reduce oxidant and inflammatory load on brain cells, but, in addition, they improve interneuronal signaling, increase neurogenesis, and enhance sequestration of insoluble toxic protein aggregates (Poulose et al., 2014).
Amyloid beta-protein (Ab) is a major component of senile plaques and cerebrovascular amyloid deposits in individuals with Alzheimer's disease. Ab is known to increase free radical production in neuronal cells, leading to oxidative stress and cell death. Selective inhibition of Ab oligomer formation provides an optimum target for Alzheimer's disease therapy. Several studies have addressed the anti-amyloidogenic activities of walnuts (Lin and Salem, 2007;Muthaiyah et al., 2011).
A walnut extract has been shown to reduce Ab-mediated cell death, decrease the release of lactate dehydrogenase (a marker of membrane damage), and reduce DNA damage as evidenced by a decrease in apoptosis and a decrease in ROS generation in a concentration-dependent manner (Muthaiyah et al., 2011). Although the majority of studies have not explored polyphenols specifically, it could be hypothesized that the anti-inflammatory and anti-oxidant potential in ellagitannins could be in part responsible for the neuro-protective effect of walnuts. It is worth mentioning that ellagic acid could reduce Ab42-induced neurotoxicity toward SH-SY5Y neuroblastoma cells (Feng et al., 2009), thus ellagitannins show a promise in preventing Ab-related neurodegenerative diseases.
Another interesting effect of ellagic acid is a potential analgesic property; different animal models of pain were used to test possible mechanisms underlying systemic antinociception after ellagic acid administration with dose-dependent analgesic effects observed (Taghi Mansouri et al., 2013;Mansouri et al., 2015).

Future trends and conclusions
In view of the experimental evidence mentioned in this review, we conclude that walnut polyphenols have obvious and numerous disease-preventive properties. The molecular functions attributed to walnut polyphenols indicative of their capacity have been performed mainly in in vitro studies, which, in order to accurately assess biological responses, must only use physiologically relevant concentrations. Therefore, bioavailability and in vivo biological efficacy are critical issues that must be correlated before drawing any conclusions on the potential health benefits of specific bioactive compounds, including polyphenols. Nonetheless, evidence suggests that incorporating walnuts into a healthy diet could aid in the prevention and modulation of several disease states. Although, this review has focused on polyphenols, several bioactive compounds in walnuts have been linked to disease prevention; as such, it is important to incorporate walnuts as a whole, to be able to reap the benefits of the variety of compounds contained in walnuts. Further research, particularly human trials, are warranted, but current evidence is encouraging enough to make walnut polyphenols bioactive compounds that should be continued to explore.