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The Beneficial Effects of Pomegranate Juice on Immunity and Intestinal Integrity in Healthy Albino Rats

Pomegranate has long been recognized as a "healing food," with significant health benefits in a variety of disorders. It has potent anti-inflammatory, antioxidant, anti-obesity, and anticancer effects, thus there has been lately a virtual outbreak of interest in the pomegranate as a functional food. Pomegranate may have physiological effects on the intestines, and the gut microbiota plays an essential part in its therapeutic effects. Therefore, we believe that the beneficial effects of pomegranate are intimately tied to the intestine. Hence, we evaluated the immunomodulatory effect and the intestinal health benefits of the administration of pomegranate juice (PJ). Rats received 1 ml of PJ for consecutive 28 days. The present data revealed that PJ administration did not affect the production of TNF-α, INF-γ, and IL-8, but it enhanced the production of IL-4 exerting an anti-inflammatory effect. It increased villus heights and goblet cell numbers. Moreover, it improved mucosal immunity by enhancing the development of germinal center (GC) in the gut-associated lymphoid tissue (GALT) that is related to the production of efficient IgA. Therefore, pomegranate juice can increase mucosal integrity, improve intestinal morphology, which beneficially alters digestion in the small intestine, and enhance mucosal immunity against pathogens.


Introduction
The importance that nutrition plays in the prevention and treatment of many illnesses has become widely recognized during the last decades (Viuda-Martos et al., 2010a), and many researchers have shown a growing interest in how food products may assist in preserving health. Recently, there is a lot of emphasis on functional foods, which, aside from their fundamental nutritional capabilities, provide physiological advantages and play a key role in illness prevention or slowing the progression of chronic diseases.
Pomegranate (Punica granatum), which belongs to the family Punicaceae, is an ancient fruit that has been consumed for thousands of years in numerous cultures. Pomegranate has long been recognized as a "healing food," with significant health benefits in a variety of disorders (Vidal et al., 2003). Indeed, in folk medicine, the pomegranate was often used to eliminate parasites, and to treat and cure ulcers, diarrhea, acidosis, dysentery, bleeding, microbiological infections, and respiratory diseases. It was also utilized as an antipyretic (Larrosa et al., 2010). Because of its multi-functionality and high usefulness in the human diet, there has been lately a virtual outbreak of interest in the pomegranate as a medical and nutritional product.
The importance of pomegranate as a functional food may be attributed to the fact that it contains a variety of compounds in various regions of the fruit with favorable physiological activities that are effective in illness risk reduction, especially with anti-oxidative (Schubert et al., 1999 andGil et al., 2000) and anti-inflammation (Shukla et al., 2008;Larrosa et al., 2010) properties. Consumption of this fruit or its juice protects against and may even improve the course of various prevalent disorders, including obesity, diabetes, cardiovascular disease, inflammatory diseases, and even some types of Middle East J. Appl. Sci., 11(4): 879-886, 2021EISSN: 2706-7947 ISSN: 2077-4613 DOI: 10.36632/mejas/2021 880 cancer (Reviewed in Viuda-Martos et al., 2010b). It has been also inferred that pomegranate may have physiological effects on the intestines (Mosele et al., 2015). It is worth noting that the gut microbiota plays an essential part in the therapeutic effects of pomegranate since their metabolites boost the pomegranate's health benefits. Accordingly, the mechanism of pomegranate's anti-inflammatory activity is closely linked to microbiota that lives in the gastrointestinal tract (Zhao et al., 2019). Taking these points into consideration, we believe that the beneficial effect of pomegranate is intimately tied to the intestine. Therefore, the objective of this study was to assess the immunomodulatory effect of pomegranate juice (PJ) on the gut by measuring particular immunological parameters such as pro-inflammatory cytokines and chemokines (TNF α, IFN,γ, and IL-8), antiinflammatory cytokines (IL-4), and immunoglobulin-A. (IgA). We also investigated its effect on the ileum by performing histological and histometrical analysis.

Materials
Adult male albino rats (Rattus norvegicus) weighing between 195 and 250 g were obtained from the breeding colony of the Ministry of Health (Helwan-Egypt). Animals were housed under normal laboratory conditions and provided food and water ad libitum. All animals used in this experiment were cared for in accordance with institutional guidelines and followed the Guide for Care and Use of Laboratory Animals. Pomegranate fruits were purchased from a well-known market (Cairo, Egypt.). Pomegranates were washed; the seeds were manually removed then crushed and squeezed by a special juice-making machine to yield concentrated fresh juice of pomegranate daily (Amal et al., 2012).

I. Experimental design
After the adaptation period, animals were randomly divided into two groups of 9 rats each: control, and Pomegranate group (PJ).
Group 1 (control): The animals fed with a normal diet. Group 2 (PJ): pomegranate juice at a dose of I ml/day was administered orally once daily for 4 weeks (Türk et al., 2008).

II. Collection of samples
At the end of the experimental period, animals were sacrificed. Blood samples were collected from the jugular vein of each rat. After incubation at room temperature, sera were collected followed by centrifugation at 4000g for 30 min and kept at -80 for immunological measurements. Samples were removed from the ileum for histological preparation.

IV. Measurement of Anti-inflammatory cytokine
A commercial kit (BOSTER Cat. No. EK0406) was used for determining IL-4 in sera as picograms per milliliter (pg/ml) according to the manufacturer's instructions.

V. Measurement of immunoglobulin-A (IgA)
IgA serum levels were quantified as nanograms per milliliter (ng/ml) using an ELISA assay kit (Abcam ab157735) according to the manufacturer's instructions.

VI. Histopathological and Histometry studies
Small portions from the ileum were fixed, sectioned at 5 μm, and stained using standard procedures Explicitly, some sections were stained with Hematoxylin and Eosin (HE) for the evaluation of the general histological architecture, and other sections were stained with Periodic Acid Schiff reagent (PAS) for the determination of goblet cell count.
GALT (gut-associated lymphatic tissue) was also investigated in ileum sections, with a special focus dedicated to the lymphatic region of individual lymphatic follicles (LF) and their various components. The lymphatic follicles were divided into their specified compartments (cortical area, germinal center, coronal region, and dome region) according to Cesta (2006). These compartment areas (μm 2 ) were measured in five LFs from each tissue sample in randomly selected fields.

Statistical analysis
SPSS 16.0 software (SPSS Inc. Chicago, IL, USA) was used for the statistical analysis. Statistical assessment was assessed by one-way ANOVA followed by Tukey test. The data were stated as mean ± SE (standard error). P-values was less than 0.05 were considered statistically significant.

Effect of PJ on Anti-inflammatory cytokine (IL-4)
PJ caused a significant increase (P < 0.05) in IL-4 serum concentrations as compared to those of the control group ( Fig. 1.B.).

Effect of PJ on Immunoglobulin-A (IgA)
Data shown in Fig. 1.C. revealed that oral administration of PJ resulted in a significant increase (P < 0.05) in the serum levels of IgA compared to their corresponding controls.

Histology and Histometry studies Ileum section
Investigation of ileum sections from control rats (Con) showed normal architecture of crypts and villi ( Fig. 2A-B). The effects of oral administration of PJ on the morphology of the ileum mucosal layer are presented in Fig. 2C-D. Specifically, administration of PJ was associated with a marked improvement in the mucosal architecture compared to the control group. Table 1 shows the effects of PJ administration on villus height after 4 weeks. The villus height was significantly increased (P < 0.05) in the PJ group compared to the control group.

Crypt depth
As shown in Table 1, there was a slight decrease in the mean crypt depths between the PJ group and the control group (P < 0.05).

Villus/crypt Ratio
After 4 weeks of PJ administration, villus/crypt ratios were elevated compared to the control group (Table 1; P < 0.05).

Discussion
The immune system is considered as a biological marker that may be used to assess the potential health benefits of dietary supplements in food for human and animal species. Pomegranate juice is high in polyphenols, tannins, and anthocyanins, as well as vitamins C, E, coenzyme Q10, and lipoic acid (Vroegrijk et al., 2011). Its major antioxidative ingredients are anthocyanins and ellagic acid derivatives, which are the principal elements of the fruit's juice and give it its color (Jurenka 2008). In the current study, the administration of PJ increased the levels of the anti-inflammatory cytokine . This is in accordance with Oliveira et al., (2010) who recorded a significant increase in the antiinflammatory cytokine (IL-4) in healthy animals after pomegranate intake. Additionally, many researchers have found that pomegranate fruit has anti-inflammatory qualities (Ahmed et al., 2005, Lansky andNewman 2007;Shukla et al., 2008;Larrosa et al., 2010). The mechanism by which the pomegranate exerts its anti-inflammatory effect is through the inhibition of cyclooxygenase (COX) and lipooxygenase (LOX), which are crucial enzymes in the conversion of arachidonic acid to prostaglandins and leukotrienes that are important inflammatory mediators (Ardekani et. al., 2011).
Our data indicated that pomegranate administration resulted in a marked increase in the mucosal integrity and clarity by increasing ileal villus height and the number of goblet cells and slightly decrease the crypt depth. The goblet cells secrete mucin which protects the gastrointestinal membrane from toxic  Appl. Sci., 11(4): 879-886, 2021EISSN: 2706-7947 ISSN: 2077-4613 DOI: 10.36632/mejas/2021 food agents and pathogenic bacteria (Kindon et al., 1995). It is well known that the heights of the intestinal villi correspond with the epithelial cells; specifically greater villi indicate more mature epithelial cells and higher intestinal absorption efficiency (He, 2006). Increased villus height can be caused by an increase in crypt cell proliferation combined with a decrease in apoptosis. The quantity of crypt cells with the ability to divide represents the intestinal membrane's growth condition. Changes in crypt depth express the rate of crypt cell division and can influence digestion in the small intestine (Kato et al., 1999). Pomegranate as a rich source of antioxidant polyphenols exhibits gastrointestinal protective activity through the accumulation of large amounts of polyphenol metabolites in the intestine, which exhibits prebiotic properties (Li et al., 2015). The same authors reported that both pomegranate extract and pomegranate juice enhanced the growth of lactobacilli and bifidobacteria while inhibiting the growth of clostridia and Enterobacteriaceae.
The present study showed that administration of PJ increased mucosal immunity by boosting the development of germinal center (GC) of payer's patches and increased the production of IgA. Previous studies indicated that microbiota and/or dietary antigens affect the properties of GC in PPs via differentiation of follicular helper T cells to produce efficient IgA, which is essential in immune protection against pathogens (Macpherson et al., 2000;Hara et al., 2019).
Conclusively, based on the current findings, it is interesting to confer that pomegranate juice may regulate the health by improving mucosal integrity and intestinal function, which beneficially can alter digestion in the small intestine, and enhance mucosal immunity against pathogens without any adverse side effects. As a result, the use of pomegranate juice in human nutrition can be recommended due to its favorable qualities.

Disclosure of interests
The authors declare that there are no conflicts of interest concerning this article.