Could Pomegranate Juice Help in the Control of Inflammatory Diseases?

Fruits rich in polyphenols, such as pomegranates, have been shown to have health benefits relating to their antioxidant and anti-inflammatory properties. Using data obtained from PubMed and Scopus, this article provides a brief overview of the therapeutic effects of pomegranate on chronic inflammatory diseases (CID) such as inflammatory bowel disease (IBD), rheumatoid arthritis (RA), metabolic and cardiovascular disorders, and other inflammatory-associated conditions, with an emphasis on fruit-derived juices. Most studies regarding the effects of pomegranate juice have focused on its ability to treat prostate cancer, diabetes, and atherosclerosis. However, pomegranate juice has shown therapeutic potential for many other illnesses. For instance, a small number of human clinical trials have highlighted the positive effects of pomegranate juice and extract consumption on cardiovascular health. The beneficial effects of pomegranate components have also been observed in animal models for respiratory diseases, RA, neurodegenerative disease, and hyperlipidaemia. Furthermore, there exists strong evidence from rodent models suggesting that pomegranate juice can be used to effectively treat IBD, and as an anti-inflammatory agent to treat CID. The effects of pomegranate intake should be further investigated by conducting larger and more well-defined human trials.


Introduction
Inflammation is a complex biological response to tissue injury and infection. Chronic inflammation has been shown to be involved in the onset and development of a range of disorders. Chronic inflammatory disease (CID) is a general term used for conditions where persistent inflammation plays a central role in disease pathology [1]. Examples of CID include rheumatoid arthritis (RA), inflammatory bowel disease (IBD), chronic obstructive pulmonary disease (COPD), asthma, and psoriasis. Patients with CID present with heavy infiltration of inflammatory cells at the site of disease-e.g., joints, intestinal mucosa, lungs, and skin-and show elevated levels of inflammatory mediators [2]. Dysfunctional inflammatory responses have also been implicated as contributors to other chronic diseases such as atherosclerosis, type 2 diabetes, obesity, insulin resistance, and certain neurodegenerative diseases (e.g., Alzheimer's disease) [3].
Ellagitannins (ET) and ellagic acid (EA) are polyphenols present in some fruits, nuts, and seeds-such as pomegranates, black raspberries, raspberries, strawberries, walnuts, and almonds [4]. It has been shown that ET-rich fruits have anti-oxidant, anti-inflammatory, anti-neoplastic, and chemo-preventive properties [5]. Pomegranate (Punica granatum L.) is a rich source of ET punicalagin 1), which aroused considerable interest in pomegranate fruit as a novel therapeutic within the last several years (Figure 2).  Although it is widely accepted that pomegranate intake can provide significant health benefits, the results of human clinical trials using pomegranate juice as a therapeutic agent have been inconsistent. This may, in part, be due to variability in the composition of the administered pomegranate products. Several studies have suggested that pomegranate intake has positive effects on blood pressure [6,7] and cardiovascular risk in diabetic [8,9], obese [10,11], hypertensive and ischemic patients [12,13]. Conversely, a meta-analysis found that pomegranate had no effect on lipid profiles [14]. In addition, a meta-analysis of data from five prospective trials did not find a significant effect of pomegranate juice on plasma C-reactive protein (CRP) levels [15].
No clear consensus has yet emerged on the putative anti-inflammatory effects of pomegranate intake on CID. Therefore, we conducted a systematic review to provide an overview of the evidence of the potential benefits of pomegranate products-with an emphasis on fruit-derived juices-on this occurrence.   Although it is widely accepted that pomegranate intake can provide significant health benefits, the results of human clinical trials using pomegranate juice as a therapeutic agent have been inconsistent. This may, in part, be due to variability in the composition of the administered pomegranate products. Several studies have suggested that pomegranate intake has positive effects on blood pressure [6,7] and cardiovascular risk in diabetic [8,9], obese [10,11], hypertensive and ischemic patients [12,13]. Conversely, a meta-analysis found that pomegranate had no effect on lipid profiles [14]. In addition, a meta-analysis of data from five prospective trials did not find a significant effect of pomegranate juice on plasma C-reactive protein (CRP) levels [15].
No clear consensus has yet emerged on the putative anti-inflammatory effects of pomegranate intake on CID. Therefore, we conducted a systematic review to provide an overview of the evidence of the potential benefits of pomegranate products-with an emphasis on fruit-derived juices-on this occurrence. Although it is widely accepted that pomegranate intake can provide significant health benefits, the results of human clinical trials using pomegranate juice as a therapeutic agent have been inconsistent. This may, in part, be due to variability in the composition of the administered pomegranate products. Several studies have suggested that pomegranate intake has positive effects on blood pressure [6,7] and cardiovascular risk in diabetic [8,9], obese [10,11], hypertensive and ischemic patients [12,13]. Conversely, a meta-analysis found that pomegranate had no effect on lipid profiles [14]. In addition, a meta-analysis of data from five prospective trials did not find a significant effect of pomegranate juice on plasma C-reactive protein (CRP) levels [15].
No clear consensus has yet emerged on the putative anti-inflammatory effects of pomegranate intake on CID. Therefore, we conducted a systematic review to provide an overview of the evidence of the potential benefits of pomegranate products-with an emphasis on fruit-derived juices-on this occurrence.

Search Strategy
We performed an extensive search using the PubMed and Scopus databases in April 2017. The following keywords and Medical Subject Headings (MeSH) terms were combined: "pomegranate" or "Punica granatum", "inflam*", and "disease*". We did not use language restrictions, and reviews were excluded. The search strategies were as follows: Medline search strategy (pomegranate*) OR (Punica granatum) AND inflam* AND disease* NOT review*; Scopus search strategy (TITLE-ABS-KEY (pomegranate*) OR TITLE-ABS-KEY (Punica granatum) AND TITLE-ABS-KEY (inflam*) AND TITLE-ABS-KEY (disease*)) AND (LIMIT-TO (DOCTYPE, "ar")).

Results and Discussion
The literature selection process was conducted following PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) recommendations [16]. The initial search yielded 156 hits after the exclusion of duplicates. During the screening process (reviewing of titles and abstracts), 25 records were excluded. After full-text analysis, another 76 papers were excluded. Altogether, 55 papers were selected for detailed evaluation. Both reviewers independently selected the evaluated articles. Figure 3 shows the flow chart of the selection procedure of the papers.

Search Strategy
We performed an extensive search using the PubMed and Scopus databases in April 2017. The following keywords and Medical Subject Headings (MeSH) terms were combined: "pomegranate" or "Punica granatum", "inflam*", and "disease*". We did not use language restrictions, and reviews were excluded. The search strategies were as follows: Medline search strategy (pomegranate*) OR (Punica granatum) AND inflam* AND disease* NOT review*; Scopus search strategy (TITLE-ABS-KEY (pomegranate*) OR TITLE-ABS-KEY (Punica granatum) AND TITLE-ABS-KEY (inflam*) AND TITLE-ABS-KEY (disease*)) AND (LIMIT-TO (DOCTYPE, "ar")).

Results and Discussion
The literature selection process was conducted following PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) recommendations [16]. The initial search yielded 156 hits after the exclusion of duplicates. During the screening process (reviewing of titles and abstracts), 25 records were excluded. After full-text analysis, another 76 papers were excluded. Altogether, 55 papers were selected for detailed evaluation. Both reviewers independently selected the evaluated articles. Figure 3 shows the flow chart of the selection procedure of the papers. Human clinical trials are relatively few (Table 1) and were mainly focused on the effects of pomegranate consumption on cardiovascular health.  Human clinical trials are relatively few (Table 1) and were mainly focused on the effects of pomegranate consumption on cardiovascular health.  Table 2 summarises the data published on human trials related to pomegranate intake. Unfortunately, there exists a limited number of human trials available concerning the effects of pomegranate on the outcomes of patients with CID. Within these studies, we found the tested dietary products and experimental designs to be highly variable. Only one trial has been conducted using pomegranate seed oil [32], whereas most other studies have been carried out using either pomegranate juice [33][34][35][36][37] or the commercially available whole fruit phenolic extract POMx TM [37,38,64], which are both safe and well-tolerated by CID patients. The doses of pomegranate juice or extract used also varied from study to study as groups used a range of 100 to 500 mL [35] of juice and 0.5 to 1 g of POMx TM [37,38]. Additionally, the study designs used differ for each trial-some used pre-and post-test schemes [33,34] or randomised placebo-controlled tests [32,35,36,38,64], and one trial even lacked an appropriate placebo comparator [37]. Finally, the duration of each study is not consistent-some performed acute (1 day) [33] or short-term (1 week) [35] trials and others assessed the long-term (6-12 months) effects of pomegranate intake [36,38]. While these studies focused primarily on patients affected by metabolic and cardiovascular disorders [32][33][34][35][36][37][38] and RA [64], there exist three clinical trials that are currently in progress exploring the therapeutic potential of pomegranate juice on IBD, memory impairment, ageing and skin inflammation (Table 3).

Findings Related to Pomegranate Products Consumption and CID in Humans
Even though the use of differing approaches renders it difficult to draw general conclusions, there is a generally positive effect of pomegranate consumption in patients with chronic inflammatory disorders. Pomegranate juice appears to have promising hypotensive properties in patients with hypertension [33] or metabolic syndrome [35,71], and in patients undergoing dialysis [38]. It also resulted in a slight amelioration of lipid profiles in patients with cardiovascular disease (CVD), as pomegranate intake elevated endogenous levels of high-density lipoprotein (HDL)-cholesterol [34] and reduced triglyceride (TG) levels [32]. However, several studies have been unable to confirm pomegranate's TG-and cholesterol-lowering effect [34,35,37,38]. With regards to risk factors for CVD, the consumption of 150 mL of pomegranate juice-restricted to one serving per day-did not influence the level of circulating soluble adhesion molecules or markers of atherosclerosis and subclinical coronary heart disease (CHD) in hypertensive individuals [33]. Evidence for the beneficial effects of pomegranate on CVD was obtained using long-term consumption (100 mL daily for one year) [36] or with a greater intake of juice (500 mL daily) [35].
Markers of systemic inflammation have not been consistently evaluated in many of these studies, a factor which has an impact on the conclusions drawn from each trial. It has been shown that circulating pro-inflammatory cytokines in patients affected by cardiovascular disorders are slightly reduced by the consumption of pomegranate juice and extract. Notably, a decrease in the level of interleukin 6 (IL-6)-a well-known pro-inflammatory cytokine-has been demonstrated in patients with type 2 diabetes [34] and in patients undergoing haemodialysis [36]. Conversely, pomegranate intake has no effect on the plasma levels of CRP or tumour necrosis factor α (TNF-α), except in patients with metabolic syndrome [35] or those undergoing dialysis [36]. It is generally accepted that the anti-inflammatory effects of pomegranate intake are mediated by its anti-oxidant properties. In agreement, pomegranate intake results in an improvement in plasma antioxidant capacity, as it has been shown to decrease the prevalence of oxidatively damaged molecules and increase anti-oxidant-dependent immune responses in patients affected by CID [34,64].
In conclusion, evidence from human trials indicates that pomegranate, when administered as a juice in high doses or for an extended period, can reduce oxidative stress and systemic inflammation. However, there exist limitations to many of these studies as these trials were limited in sample size, used short durations of supplementation, and/or lacked necessary controls.
Several groups have studied the effects of pomegranate on the prevention and amelioration of atherosclerosis and other CVD symptoms. de Nigris et al. (2007) [43] reported that supplementation with pomegranate juice or pomegranate fruit extract decreased the expression of vascular inflammation markers and transforming growth factor β-1 (TGFβ-1), and, likewise, increased endothelial NO synthase (eNOS) levels in a rat model of metabolic syndrome. Additionally, Labsi et al. (2016) [74] showed that intraperitoneal treatment with pomegranate peel extract for two months after the induction of echinococcosis significantly reduced the nitric oxide (NO) and TNF-α levels in Swiss albino mice. Pomegranate juice supplementation also ameliorated cardiac hypertrophy, and reduced oxidative stress and expression levels of interleukin 1β (IL-1β) and several fibrotic markers in the aorta of rats exposed to cigarette smoke [39]. Similarly, pomegranate juice supplementation slowed the development of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) in a diet-induced obesity rat model [48]. Other authors illustrated the hypoglycaemic activity of pomegranate flowers [45], seeds [46], peel [45], and juice [41,48] in addition to its bioactive compound EA [49] using various rodent models of diabetes. Here, pomegranate-derived products appeared to regulate the activation of peroxisome proliferator-activated receptor γ (PPAR-γ) [46], a known regulator of fatty acid storage and glucose metabolism.     High-fat and high-carbohydrate diet-induced metabolic syndrome ↑ retroperitoneal, epididymal, omental, and total abdominal fat, ↔ whole-body fat mass, ↓ whole-body lean mass, ↓ glycaemia, ↓ plasma TG, TC, NEFA, uric acid, urea, and CRP, ↓ plasma ALT, AST, ALP, and LDH activity, ↔ plasma albumin and bilirubin, ↓ SBD, ↑ coronary endothelial-dependent relaxation, ↔ Nrf2 protein expression in heart, ↑ Nrf2 protein expression in liver, ↓ NF-κB expression in heart and liver, ↑ CPT1 expression in heart and liver  High-fat diet-induced NAFLD ↓ body weight gain, ↓ serum TG, HDL-C, and LDL-C, ↔ serum C, ↓ serum insulin, leptin, and adiponectin, ↓ HOMA-IR, ↓ serum ALT level, ↓ liver tissue weight, ↓ hepatic TG and TC, ↓ expression of SREBP-1c precursor protein, ↔ expression of SREBP-1c mature protein, expression of FA biosynthesis-related genes (↓ SREBP-1c, ↓ FAS, ↓ ACC1, ↓ SCD1), expression of TG biosynthesis-related genes (↓ ACLY, ↔ GPAM, ↑ DGAT-1 and-2), ↓ serum CRP level, IL-1β, IL-4, IL-6, and TNFα, ↓ serum IgA, IgG, and IgM, ↓ protein carbonyl content in liver tissue and liver mitochondria, ↓ lipid peroxidation in liver, ↑ hepatic total SOD activity, ↓ hepatic GSH and GSSG levels, ↑ GSH/GSSG ratio, ↓ Nrf2, HO-1, NQO-1, and UCP2 protein expression in liver, ↑ ATP content in liver, ↑ activities of mitochondrial complexes I, II, and IV in liver, ↑ expression of genes PGC-1-α and PPAR-α in liver, ↔ expression of PGC-1β gene in liver, ↑ PGC-1α protein expression in liver, ↑ expression of genes CPT1A, CPT1B, and ACAD in liver  ↑ behavioural performance, ↓ TNF-α in spleen and brain, ↓ NFATc1 activation in spleen and brain, ↑ p-NFATc2/NFATc2 ratio in brain, ↓ p-IκB/IκB ratio in brain, ↓ plaques in brain     Several studies assessed the efficacy of pomegranate fruit as an antiproliferative agent in animal models of prostate hyperplasia and carcinoma. Malik and Mukhtar (2005) [69] demonstrated that, in vivo, oral administration of pomegranate fruit extract results in tumour growth inhibition accompanied by a reduction in serum prostate-specific antigen (PSA) levels. The decreasing of serum PSA levels have also been confirmed in two studies in humans [75,76]. In addition, dietary supplementation of 4% pomegranate extract with a standard chow diet inhibited neuro-inflammation in a transgenic mouse model of Alzheimer's disease (AD). A delay in the formation of senile plaques and the loss of synaptic proteins was also observed [55,56]. Conversely, treatment with pomegranate juice did not protect neuronal degeneration in a separate study that used a rat model of AD, but instead exacerbated neuronal cell death and inflammation [60].
In conclusion, these studies using animal models of inflammation-related disease offer a crucial step in answering phenomenological questions related to the pathology of CID, but cannot be fully applied clinically due to the relatively high doses of pomegranate products used.

In Vitro Anti-Inflammatory Activity of Pomegranate Extracts or Pomegranate-Derived Bioactive Compounds
The anti-inflammatory effects observed in vivo have generally been confirmed in vitro. However, experimental conditions applied to cell cultures also tend to vary considerably with regards to the concentration of pomegranate extracts and time-points used ( Table 5). In vitro, several pomegranate products have been tested including whole juice [61,63,70], extracts from the husk [29], seeds [62], or pulp [61,77], bioactive compounds present in pomegranate juice (ET [29,53,77] and EA [30]), or POMx TM extract [31]. Of all the groups working with these compounds, only Giménez-Bastida et al. (2012) [28] decided to assess the effects in intestinal cells of EA, which is present at high concentrations in the human colon. It is well known that pomegranate ETs are first hydrolysed to EA followed by transformation into the metabolite-urolithin-in the gut [5]. The importance of identifying bioactive metabolites using cell-based experimentation has been recently elaborated upon by Aragonès et al. (2017) [78].
As mentioned above, in vivo studies have defined a clear role for NF-κB in the modulation of inflammation by pomegranate extracts, a finding that appears to be confirmed in vitro. Pomegranate juice [63], POMx TM extract [31], and their bioactive compounds-punicalagin [53] or delphinidin [66]-all suppressed NF-κB activation in various types of cells. It was found that ET reduced the expression of NF-κB target genes, including IL-6 and interleukin 8 (IL-8), upon exposure to pro-inflammatory stimuli in intestinal cells [29], while EA [30] and POMx TM [31] reduced NF-κB activation in various subsets of immune cells, and anthocyanin delphinidin reduced inflammation in rheumatoid arthritis cells [66]. Taken together, these results suggest that ET and other bioactive compounds present in pomegranate juice show anti-inflammatory effects in vitro, and that the mechanisms involved appear to be related to inactivation of NF-κB signalling.  Neuronal cells

Conclusions and Future Directions
Despite abundant literature on the putative effects of pomegranate fruit or extract on CID and other inflammation-related diseases, a definitive relationship between the consumption of pomegranate products and its beneficial properties has not yet been established. It is likely that the effects are due to the ingestion of pomegranate's bioactive polyphenolic molecules. To date, most scientific research on the promising health benefits of pomegranate have been carried out in animal or cell culture models. Clinical trials are currently being conducted to examine a wide range of the potential health effects of pomegranate. However, these trials are few. The most promising properties of pomegranate thus far are related to its effects on diabetes, metabolic syndrome, and cardiovascular diseases. Further studies are required to determine the specific effects of ET-containing foods [80], and to explain the health benefits of pomegranate on CID.