*5.2.2 Chemoprevention of cancers*

*Tannins - Structural Properties, Biological Properties and Current Knowledge*

**5.1 Effect of ETs and EA in GI tract**

**5.2 Effect of urolithins**

*5.2.1 Anti-inflammatory effect*

within the GI tract, as these compounds are poorly bioavailable. On the other hand, urolithin production is mediated by microbiota and studies on animals can hardly be extrapolated to humans, except in the case of germ-free animals used in human microbiota-related researches. Therefore, in this review, even though research studies are much more scarce, we will report results of biological activities of ETs and EA only related with the GI tract, and for urolithins, given the importance of microbiota in their metabolism, we will focus only on the results of clinical trials with humans.

In in vitro model of colon cancer, ellagic acid was found to have a significant antiproliferative effect inducing apoptosis of cancer Caco-2 cells via a mitochondrial pathway and without side effects on normal colon cells [55]. In another study in rats, EA showed anti-inflammatory properties by iNOS, COX-2, TNF-α, and IL-6 downregulation due to NF-κB repression. Authors conclude that EA may exert a chemopreventive effect on colon carcinogenesis [56]. Furthermore, ETs and EA have a high antioxidant activity (even higher than urolithins) and could be highly efficient to scavenge oxygen free radicals and eventually prevent inflammation and colon cancer [21]. At last, in nasopharyngeal carcinoma cell lines (NPC-BM1), EA has showed ability to downregulate Bcl-2 and DNA fragmentation, by increasing

Numerous studies have demonstrated the metabolism of EA into urolithins, in approximately 12–24 h, with persistence of urolithins up to 4–7 days in urine after dietary intervention [9, 41, 42, 58], and research interest has shifted to the potential effect of urolithins on health. Actually, urolithins may exert a much more consistent effect at the systemic level than EA with concentration in body fluids at least one order of magnitude higher in body fluids. Actually, EA has been reported in plasma at concentration around some nanomoles per liter, while urolithins have been

Inflammation is a primary defensive response against harmful factors that involved different mechanisms including the immune system cells [8]. The impact of urolithins on inflammatory processes has been well established on various in vivo and in vitro models [59–61]. In in vitro models, urolithin aglycones have been tested, while in vivo glucuronide conjugates of urolithins are the predominant metabolites present in plasma, tissues, and urine [62]. Urolithin aglycone is highly bioactive against inflammation, but some authors suggest that urolithin conjugate may be even more active. In a study, in which urolithin conjugates (iso-Uro-A-gluc, Uro-A-gluc, and Uro-B-gluc) were isolated from urine of a volunteer after ingestion of pomegranate juice (0.5 L/day), walnuts (30 g/day), hazelnuts (30 g/day), and fresh raspberries (200 g/day) for 5 days, the cleavage of glucuronides by endogenous β-glucuronidases released by human neutrophils was observed . β-Glucuronidase is an enzyme that is released from inflammatory cells and the lysosomes of necrotic cells, and high levels can be found in most solid tumors. Therefore, a wide number of structurally diverse glucuronide prodrugs have been designed with the aim of enhancing the selectivity of cancer chemotherapy. The results suggest that the selective activation of urolithin glucuronides by β-glucuronidase could locally increase the concentration of bioactive urolithin aglycones. More clinical trials are needed to

caspase-3 enzymatic activity, which reduces telomerase activity [57].

reported at concentration level up to 80 μmol/l [58].

**30**

Several in vitro and in vivo (animal or human) studies have reported a protective effect of urolithins on prostate cancer [64–66]. However, recent clinical interventions have pointed out inconsistent results. A recent review compiles the data of clinical trial studies after consumption of pomegranate juice or extracts and discusses whether urolithin could inhibit or slow the growth of prostate cancer in patients. The authors reported a significant increase in urolithin A in prostate but a nonsignificant reduction in 8-hydroxy-2-deoxyguanosine, a marker of oxidation in cancer tissue, for neoadjuvant patients subjected to radical prostatectomy after pomegranate intake (1200 mg polyphenols/day) compared with placebo in a large trial (4 weeks, n = 33) [67]. Similar results with muscadine grape skin extract have evidenced no benefit on recurrent prostate cancer patients in spite of urolithin A increase [68]. Nonetheless, authors [67] have noted in a specific group of patients (named AA genotype), which has been previously associated with more hostile prostate cancer and more sensitivity to antioxidants, a significant increase in a prostate-specific antigen doubling times (PSADT), an antigen that is claimed to slow tumor growth.

After an acute intake of grumixama cherry juice (Brazilian) by healthy women (n = 10), the antiproliferative activity of urolithins against breast cancer cells (MDA-MB-231) was evaluated. The extracts of urine exhibited seven urolithins, mainly urolithin C and urolithin A. Those extracts obtained during 2–4 h after juice intake presented the highest inhibition of proliferation of MDA-MB 231 breast cancer cells. This inhibition was attributed to a significant G2/M cell cycle arrest (apoptosis) occurred in MDA-MB-231 cells, and was demonstrated by the increase in sub-G0/ G1 populations. Additionally, the authors linked this inhibition to a possible synergy among anthocyanins and urolithins [12]. Then, the modulation of the positiveestrogen receptor in breast cancer cells (MCF7) is probably one of the potential actions of urolithin conjugates. On the other hand, in a study realized with breast cancer patients (n = 19) who consumed a mixed extract (493.4 mg phenolics/day) containing pomegranate, orange, lemon, olive, cocoa, grape seed extracts plus resveratrol, theobromine, and caffeine, it was shown that the main metabolites detected in breast tissues were urolithin-A-3-O-glucuronide. Nonetheless, no antiproliferative or estrogenic/antiestrogenic activities in MCF-7 breast cancer cells were reported [69].

Ellagitannin gut microbiota-derived metabolites have shown a wide range of colon anticancer effects both in cellular and animal studies [70–72]. However, the current clinical evidence that confirms their colorectal cancer (CRC) chemopreventive effect in humans is still very weak. A study evaluated the modification of microRNAs (miRs) expression, one CRC biomarker, in normal and malignant colonic tissues from CRC patients after pomegranate extract intake (900 mg/day before surgery). As a result, pomegranate consumption seems to moderate the modulation of various specific miRs in colon tissue, but there was no association between tissue urolithins and the detected miRs changes, which were attributed to a possibly critical surgery alteration in miRs levels that did not allow to discriminate between malignant and normal tissues [73]. Another more recent study in 35 patients with colorectal cancer (CRC), daily supplemented with pomegranate extracts, was conducted to evaluate the expression of various CRC-related genes in normal and cancerous colon tissues. Before (biopsies) and posterior (surgical samples) to pomegranate intake (5-35 days). Despite the consumption of pomegranate extract was significantly associated with a balancing effect in the expression of genes regulated by the experimental protocol, these results were not associated with the individual metabotypes or the levels of urolithins and EA in the colon tissues. Consequently, the in vitro effects were not reproduced in vivo evidencing discrepancy between results [70]. In general, we can conclude that there is a lack of clinical interventions with ET-rich food in humans; besides, these kinds of studies are essential to corroborate the real effect of urolithins on cancer.

#### *5.2.3 Reducing risks of cardiovascular disease (CVD)*

The gut microbiota is frequently presented as a key factor in the evolution of obesity and cardiovascular disorders (CVD) [74]. One clinical study clustered urolithin metabotypes (UMs) of 18 healthy overweight/obese subjects with the aim of correlating metabotype status with CVD biomarkers after pomegranate extract consumption. In baseline and before UM clustering, the whole group exhibited mild dyslipidemia, and after clustering, only the serum lipid profile of UM-B individuals (n = 15) showed moderate risk values in total cholesterol, intermediate-LDLcholesterol, as well as other serum lipids related to CVD risk. After ET intervention, only blood biomarkers of UM-B subjects were improved after pomegranate extract intake, reducing their CVD risk. Interestingly, a dose-dependent behavior was notable only in UM-B patients [48].

Another experiment comparing healthy patients with patients with metabolic syndrome (MetS), both consuming walnuts, showed that urolithin A only was inversely correlated with glycaemia in MetS individuals. Additionally, when MetS patients with UM-A were treated with statin, their lipid profile became similar to healthy individuals. This was not the case for individuals with UM-B [74].

Another study showed that the increasing relative importance within the microbiota of bacteria from the Coriobacteriaceae family such as *Olsenella, Senegalimassilia, and Slackia*, which characterized UM-B status, was positively correlated with blood cholesterol levels and normal BMI [49].

Endothelial dysfunction and inflammation are both usual events that occur in the development of atherosclerosis. The correlation between the plasma urolithin metabolites and improvement in endothelial function after red raspberry intake was reported. Endothelial function measured as flow media vasodilation (FMD) presented two peaks, first at 1–2 h after intake, linked with EA plasma peak concentration, and second peak at 24 h, associated with urolithin-3-glucuronide and urolithin-A-sulfate absorption peaks. Similar results were reported by other authors in cranberry and blueberry juice interventions [75], but it was shown that effect was the same when consuming 200 or 400 g of raspberries. Additional distinctive key factors in atherosclerosis development have been reported, as the capacity of monocytes to adhere to endothelial cells and the uptake and efflux of cholesterol by macrophages. In vitro, urolithins and EA were able to reduce adhesion of THP-1 monocytes to human umbilical vein endothelial cells and reduce secretion of sVCAM-1 and IL-6, a cellular adhesion molecule and a pro-inflammation cytokine, respectively. Also, urolithin C and EA were associated with decreased accumulation of cholesterol in THP-1–derived macrophages [76]. Attenuation of THP-1 also was reported in the presence of urolithin A in endothelial cells and also reduced considerably the expressions of ICAM-1 and MCP-1, an intercellular adhesion molecule and a monocyte chemotactic protein, respectively [77].

#### **6. Concluding remarks**

Ellagitannins are present in considerable amounts only in some specific food sources such as berries and nuts, but some tropical fruits deserve attention. Their

**33**

*Food Ellagitannins: Structure, Metabolomic Fate, and Biological Properties*

diverse structure can be modified during food processing resulting in free ET and EA derivatives, which are poorly bioavailable. After ingestion, most ETs are spontaneously converted into EA, which is poorly bioavailable and can be used as substrate by gut microbiota. The main products resulting from the action of gut microbiota on EA are urolithins. The main biomarkers in blood and urine of ET-rich food exposure are urolithins A and B. Nonetheless, there is an important interindividual variability in the excretion of urolithins, and this observation has led to the classification of the population in three metabotypes: the "low urolithin" excreters that represent approximately 10% of the population; the "urolithin A" excreters, the most important group with approximately 55% of individuals; and finally, the "urolithin A and B excreters" that represent around 35% of the population. The metabotype status appears to be quite persistent although it can change during life span and the constant exposure to ET-rich food appears to increase urolithin production. Microorganisms from the Coriobacteriaceae family were identified as urolithin producers and the relative importance of this family within the microbiota was apparently correlated with the metabotype. The stratification of individuals by their metabotype was essential to overcome inconsistencies during clinical trial, and it must be taken into account in all future intervention studies. The positive biological effects of ETs and EA at the level of the GI tract are consistent and reported by various authors. For urolithins, the panorama is more confused, and more long-term clinical intervention studies with human are required. Nonetheless, at the end of this review, the potential health effect of ET-rich foods is definitely promising and they deserve to be part of a healthy diet as functional foods.

This publication was jointly financed by the International Research Center in Agronomy for Development (CIRAD), Montpellier, France, and the Colombian Research Cooperation in Agronomy (AGROSAVIA), Colombia. Karen Johana Ortega is a PhD student with a scholarship granted by the Colombian government

*DOI: http://dx.doi.org/10.5772/intechopen.86420*

**Acknowledgements**

**Conflict of interest**

through Colciencias 727-2015 announcement.

The authors confirm that there is no conflict of interest.

*Food Ellagitannins: Structure, Metabolomic Fate, and Biological Properties DOI: http://dx.doi.org/10.5772/intechopen.86420*

diverse structure can be modified during food processing resulting in free ET and EA derivatives, which are poorly bioavailable. After ingestion, most ETs are spontaneously converted into EA, which is poorly bioavailable and can be used as substrate by gut microbiota. The main products resulting from the action of gut microbiota on EA are urolithins. The main biomarkers in blood and urine of ET-rich food exposure are urolithins A and B. Nonetheless, there is an important interindividual variability in the excretion of urolithins, and this observation has led to the classification of the population in three metabotypes: the "low urolithin" excreters that represent approximately 10% of the population; the "urolithin A" excreters, the most important group with approximately 55% of individuals; and finally, the "urolithin A and B excreters" that represent around 35% of the population. The metabotype status appears to be quite persistent although it can change during life span and the constant exposure to ET-rich food appears to increase urolithin production. Microorganisms from the Coriobacteriaceae family were identified as urolithin producers and the relative importance of this family within the microbiota was apparently correlated with the metabotype. The stratification of individuals by their metabotype was essential to overcome inconsistencies during clinical trial, and it must be taken into account in all future intervention studies. The positive biological effects of ETs and EA at the level of the GI tract are consistent and reported by various authors. For urolithins, the panorama is more confused, and more long-term clinical intervention studies with human are required. Nonetheless, at the end of this review, the potential health effect of ET-rich foods is definitely promising and they deserve to be part of a healthy diet as functional foods.
