Pomegranate juice to reduce fecal calprotectin levels in inflammatory bowel disease patients with a high risk of clinical relapse: Study protocol for a randomized controlled trial

Inflammatory bowel disease (IBD) has a multifactorial etiology and is thought to be related to a combination of individual genetic susceptibility and environmental triggers that stimulate an inflammatory response. Ulcerative colitis (UC) and Crohn’s disease (CD), the two main types of IBD, are chronic conditions that usually follow a relapsing clinical course with phases of remission that alternate with periods of active inflammation [1]. Generally, after medically induced remission, about half of the patients who do not receive therapy will relapse in the following year [2].

Fecal calprotectin (FC), a calcium-binding protein derived from neutrophils, monocytes, and reactive macrophages [3], has been shown to be a useful marker for defining subclinical persistent mucosal inflammation in IBD and has demonstrated a positive linear correlation with both endoscopic and histological activity [4‐8]. In particular, a FC ≥ 100 μg/g intercepts a subgroup of patients in clinical remission with a high risk of relapse [9‐11]. However, a combination of this FC marker and other inflammatory response markers and disease-specific activity indices can increase diagnostic accuracy in reference to FC elevation [12‐14].

Several relevant molecular mechanisms contribute to chronic inflammation in IBD. The involved mechanisms appear to be related to the dysregulation of selected signaling pathways in the colon, such as nuclear factor κ light-chain-enhancer of activated B cells (NF-κB), c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and signal transducer and activator of transcription 3 (STAT3) phosphorylation [15, 16]. In addition, relevant molecular processes associated with clinically quiescent inflammatory states in tissues other than the colon can be studied by analyzing peripheral blood gene expression patterns to identify novel transcriptional signatures related to incoming relapses [17].

Critical goals for IBD are to preserve a condition of clinical remission. Diet may be an important factor in the maintenance of long-term diminution of disease severity. In particular, a diet rich in phytochemicals, such as polyphenols, could represent a strategy in the modulation of chronic intestinal inflammation [18]. In particular, strong evidence indicates that polyphenol-rich pomegranate supplementation exerts anti-inflammatory effects that may ameliorate the symptoms of chronic inflammatory diseases and IBD in rodent models [15], probably due to the formation of ellagitannin (ET) metabolites in the colon. ETs are polyphenols present in berries (cloudberry, raspberry, rosehip, strawberry, and sea buckthorn), pomegranate, walnuts and some other nuts, and oak-aged wines [19]. Upon hydrolysis in the gut, ETs undergo lactonization to produce ellagic acid, which is converted into urolithins by the microbiota [20]. These metabolites occur at relatively high concentrations in the colon (6–168 μM urolithin A, the most abundant metabolite, in rodents and pigs) [21] and can exert anti-inflammatory effects at both local (colon) and systemic levels [22‐25]. Despite these promising premises, few studies have explored the potential benefits of ET-rich food on IBD in humans.

In the present exploratory study, patients with IBD in stable clinical remission with increasing inflammation identified by increased FC values were asked to consume an ET-rich pomegranate juice in order to test systemic and mucosal changes in biochemical and molecular inflammatory response markers relative to a control group receiving a placebo.

Currently, there is an increased incidence of IBD, such as UC and CD. IBD clinical symptoms (diarrhea, abdominal pain, and gastrointestinal bleeding) are characterized by relapses and remissions; a severe long-term complication of chronic inflammation is the development of colorectal cancer [56]. First-line therapies in IBD are medications (e.g., 5-aminosalicylates, corticosteroids, immunomodulators, and biologics), which can have severe adverse side effects. The maintenance of IBD remission is a clinical challenge and a relatively neglected area of current research. Several authors have indicated the importance of diet for managing the course of IBD in patients, demonstrating the need for more intervention trials [57, 58].

A strong evidence base suggests that pomegranate fruit exerts anti-inflammatory effects that may alleviate the symptoms of IBD; colon tissue damage, antioxidant status, and inflammation were all ameliorated by pomegranate fruit supplementation in rodent models of IBD [22‐25]. Since pomegranate bioactive molecules, mainly ETs, are poorly absorbed in the stomach and small intestine and metabolized mainly in the colon to ellagic acid and urolithins, where they can be found at relatively high concentrations, their effects on intestinal inflammation merit attention. Some work has described the beneficial effects of pomegranate ET metabolites on inflammation in cultured colon fibroblasts [59, 60]. To date, however, no clinical trials in humans have tested the possible role of the consumption of pomegranate juice in the reduction of inflammation in IBD patients with a high risk of relapse.

The effects of the consumption of pomegranate juice in the active group are compared to those of the consumption of a placebo, which has comparable calories and sugar content, in the control group. The use of beverages as an intervention appears to be robustly acceptable and feasible [61]; in addition, the consumption of 100% fruit juice is not associated with an increased risk of diabetes [62]. Consumption of ET-containing foodstuffs and supplements is restricted throughout the trial to standardize the ET daily intake and to minimize influences of other ET sources on the study outcomes. Adherence to dietary restrictions before sample collection is assessed by food diaries, whereas FFQs are used to evaluate exposure to dietary antioxidants over time [35].

Interindividual variability in the production of different ET-derived metabolites [63, 64] is assessed in plasma and urine samples to more clearly define the effects of pomegranate juice and provide a scientific rationale for its consumption. In addition to clinical, biochemical, and endoscopic evaluations, in-depth mechanistic analyses are necessary to provide scientific evidence for the actual cause–effect relationship between pomegranate juice consumption and its possible benefits against inflammatory conditions at both colon and systemic levels. To date, very few trials have studied the associations between nutritional exposure to ET-rich foods and gene expression changes in human tissues of IBD patients [65, 66]. Some investigations were performed in the colon tissues of patients affected by colorectal cancer upon supplementation with pomegranate extracts [67‐69].

In conclusion, IBD arises from inappropriate activation of the mucosal immune system, resulting in a state of chronic inflammation. This study is an attempt to bridge the gap between pomegranate consumption and potential amelioration of the inflammatory response. The possibility of preventing relapses or at least minimizing their severity with a dietary intervention would greatly help both physicians and IBD patients.

Recruitment started in December 2016, using protocol version 2 released on July 18, 2016; it is concluded in January 2019.