Research articleAnti-inflammatory properties of a pomegranate extract and its metabolite urolithin-A in a colitis rat model and the effect of colon inflammation on phenolic metabolism☆
Introduction
Nowadays, there is an increased incidence of acute colon inflammation episodes that can be elicited by viral or bacterial infection, stress, or allergy to some peptides. Chronic inflammation bowel diseases (IBDs) like ulcerative colitis and Crohn disease have also risen in the last few years [1]. Several genes that are up-regulated in colon inflammation remain elevated in colonic cancer, and oxidative stress that accompanies chronic inflammation also contributes to the trigger of dysplasia [2].
The etiology of IBDs is not well understood, but two broad hypotheses have arisen regarding the fundamental nature of the pathogenesis of IBDs. The first contends that primary deregulation of the mucosal immune system leads to excessive immunologic responses to normal microbiota. The second one suggests that changes in the composition of gut microbiota and deranged epithelial barrier function elicit pathologic responses from the normal mucosal immune system [3].
Since ancient times, pomegranate has been regarded as a “healing food” with numerous beneficial effects on several diseases. Indeed, pomegranate has been used as an anti-helmintic and vermifuge and to cure aphtae, ulcers and diarrhea in folk medicine. The studies carried out today indicate that our ancestors were not entirely wrong and that some of properties of pomegranate were not unfounded. In vitro and in vivo studies have demonstrated antioxidant, anti-inflammatory and anticancer properties of pomegranate. Administration of 50 mg/kg of pomegranate peel extract for 28 days to rats with liver fibrosis decreased malondialdehyde (MDA) levels and myeloperoxidase activity as well as TNF-α and IL-1β levels [4]. Also, pomegranate juice, as a substitute for drinking water for 1 month, reduced basal levels of hepatic oxidative stress, decreasing DNA damage [5]. In a model of rheumatoid arthritis, pretreatment with 13.6 mg/kg of pomegranate extract (PE) decreased the arthritis incidence and IL-6 and IL-1β levels in arthritic joints [6]. Nevertheless, these studies did not take into account that when pomegranate compounds are ingested, only a fraction is absorbed. In fact, our group demonstrated for the first time that ellagitannins from pomegranate and other sources are metabolized to urolithins by colonic microbiota [7], [8], [9], [10]. These metabolites are subsequently absorbed and reach a number of organs [10]. Urolithin properties have not been deeply studied. We have previously reported the anti-estrogenic/estrogenic activity of urolithins due to their structural analogy to estrogens [11] as well as their antiproliferative activity due to the modulation of gene expression including MAPK signaling pathways [12]. Other authors have reported that ellagic acid and several synthesized urolithins inhibited the growth of human prostate cancer cells in vitro [13]. In addition, an ellagitannin-rich PE has been shown to inhibit angiogenesis in prostate cancer in vitro and in vivo, suggesting that ellagitannins are the most active fraction responsible of the effect [14]. Therefore, pomegranate properties could be mediated by the in vivo produced metabolites in addition to the original phenolic compounds present in the food matrix. Human subjects can be divided into high and low urolithin producers [15]. Whether a high or low urolithin production is beneficial or indifferent remains unanswered so far.
There is increased evidence regarding the involvement of gut microbiota on the health-beneficial effects of foods. For example, Russell et al. [16] have suggested that blueberry phenolic transformations by gut microbiota determine the action of these compounds inhibiting prostanoid production.
In view of the antioxidant, anticancer and anti-inflammatory properties of pomegranate phenolics and/or its derived metabolites, we could hypothesize that PE and/or their derived metabolites could have a beneficial effect on colon inflammation. Our aim was to evaluate the effects of diets supplemented with PE and urolithin-A (UROA) (the main in vivo-derived metabolite) in a dextran sodium sulfate (DSS)-induced colon inflammation rat model (a well-known model that mimics an inflammatory bowel disease). The underlying objectives were to assess whether the effects are due to the ellagitannins or to their microbiota-derived urolithins and to check the effect of inflammation on phenolic metabolism. For this purpose, colon tissue damage, microbiota changes, reactive oxygen species (ROS), gene expression profile, polyphenol metabolism as well as a number of inflammatory markers including prostaglandin and nitric oxide production were explored.
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Animals, diets and experimental design
Experiments followed a protocol approved by the local animal ethics committee and the local government. All experiments achieved were in accordance with the recommendations of the European Union regarding animal experimentation (Directive of the European Council 86/609/EC). Male Fischer rats (n=32) with weights ranging from 175 to 200 g were provided by the Animal Centre of the University of Murcia (Spain). Animals were randomly assigned to four groups (n=8 rats per group). Each group was
Hematology
The DSS-treated rats showed a significant decrease (P<.05) in the hematological parameters RBC count, Hb and HCT compared to control rats (Table 1). These reductions were significantly attenuated in the DSS-PE group (P<.05). The effect in the DSS-UROA group was stronger than in the DSS-PE group as both Hb and HCT were not reduced upon DSS treatment and even a slight increase was observed for RBC count (Table 1). No effect was observed in the rest of the hematological parameters analyzed.
Histological analyses
Discussion
Inflammatory bowel diseases are characterized by high levels of ROS that are produced by neutrophils and macrophages recruited in the inflamed tissue, as well as a decreased antioxidant capacity of plasma. Moreover, cytokines like TNF-α and IL-1β can also induce ROS production [24]. According to a previous study carried out with the DSS model [25], an increase in lipid peroxidation levels in colon mucosa and a decrease in antioxidant capacity of plasma were observed in our study. From the two
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This work has been supported by the Projects CICYT-BFU2007-60576 and Consolider Ingenio 2010, CSD2007-00063 (Fun-C-Food). M.L. is holder of a postdoctoral grant from MCINN (Spain), M.V.S. is the holder of a I3P-postdoctoral grant from CSIC (Spain), A.G.S. is holder of a FPI predoctoral grant from MICINN and M.A.O. is the holder of a JAE-predoctoral grant from CSIC.