2.8.2. Intestinal Regulation

Pomegranate derivative intake leads to a considerable accumulation of ellagitannins in the large intestine, where they react with the intestinal microflora [139]. Bialonska et al. [140] reported that the beneficial e ffects on microflora are mainly due to PoPx. A well-known cause of intestinal damage is represented by high-intensity exercise. In order to overcome this risk, Chaves et al. studied the consequences of the assumption of fermented milk supplemented with whey protein

(approximately 80% protein), probiotic (*Bifidobacterium animalis* subsp. lactis BB12), and pomegranate juice (*Punica granatum* L.) on the physical performance, intestinal motility and villi structure, inflammatory markers, and intestinal microbiota of in vivo model Wistar rats under high-intensity acute exercise. The group only subjected to exercise went through changes in the intestinal villi interspace, in the proportion of *Lactobacillus* species and an increase in *Clostridium* species, as well as a decrease in intestinal motility. The treated group, instead, ameliorated intestinal motility and preserved the intestinal villi interspace and the natural microbiota proportions, but the physical performance was not enhanced [141].

In 2019, George et al. demonstrated the capability of pomegranate peel extract to constrain the pathogenicity of *Citrobacter rodentium (Cr)* infections. The results obtained underlined a discrepancy between mice treated with pomegranate peel extract and the control group regarding the composition of the microbiome. Indeed, in treated animals, a reduced proportion of Firmicutes/Bacteroidetes was found by increasing Bacteroidetes and decreasing Firmicutes amounts, which led to a large reduction in *Lactobacillus.* These alterations have led to the belief that the intake of pomegranate could play a protective function against various intestinal infections [142].

Similar findings have been evidenced by Smith et al. which assessed that the treatment pomegranate peel extract was able to reduce Cr infections, weight loss, and mortality with respect to whom assumed just water as control groups. Instead, the treatment did not alter Cr colonization of the colon, whereas it reduced the colonization of the spleen. Moreover, it lowered the degree of Cr-induced colon damage, associated with mortality [143].

Nevertheless, the advances made to understand the capability of pomegranate to influence the microbiota remain limited still and need to be deepened in vivo.

#### 2.8.3. E ffects on the Male Reproductive System

According to Türk et al. [144], the consumption of pomegranate juice produces an increase in the concentration of sperm in the epididymis, higher sperm mobility and density, and a reduction of poor quality sperm compared to the reference or control group. In a more recent study, this same group of researchers suggested that ellagic acid (8) has a protective e ffect on both testicles and sperm. This e ffect may be connected to the powerful action of ellagic acid against oxidative stress [145]. Concerning erectile dysfunction or impotence, the continued inability to achieve or maintain an erection su fficient to maintain satisfactory sexual intercourse, Forest et al. [146] stated that after four weeks of treatment with pomegranate juice, patients showed better erectile activity than others who had received a product with a placebo e ffect.

However, this field the knowledge is still limited, and other studies are necessary to assess the veracity of this hypothesis.

### 2.8.4. Antidiarrheal E ffects

Qnais et al. [147] assessed the antidiarrheal e ffects of the water extract of pomegranate fruit skin on rats. The outcomes demonstrated that the extract dose-dependently inhibits spontaneous ileum movements and attenuates acetylcholine-induced contractions.

In other studies, the antidiarrheal e ffects of fruit skin on rats were evaluated by administering an oral dose of 400 mg/kg. The findings showed that pomegranate extract decreases the number of defecations and stool weight [148].

These previous findings were also supported by Zhao et al. in 2018 by studying the aqueous extract of pomegranate peels and, in particular, its bioactivity-guided fractions and bioactive components. The fraction considered responsible of the antidiarrheal activity was the ethyl acetate one, mainly composed by punicalagin, corilagin, and ellagic acid. Data also revealed that the administration of the ethyl acetate fraction at 100, 200, and 400 mg/kg was able to decrease gastrointestinal transit in charcoal meal tests in mice as well as inhibit castor oil-induced enteropooling compared to control

animals. From histopathological evaluations, it emerged that small intestine lesions of mice treated with the ethyl acetate fraction were relieved in comparison to those in mice treated with castor oil [149].

Although the studies in this sector are insufficient, they sugges<sup>t</sup> the potentiality of pomegranate in regulating intestinal motility.

### 2.8.5. Effects on Oral Health

Currently, science recognizes that chronic periodontal inflammatory disorder is strictly linked to the worsening of cardiovascular disease [150].

DiSilvestro et al. [151] have demonstrated that an oral rinse based on extracts of pomegranate would significantly reduce (about 84%) the number of microorganisms from the dental plaque.

Sastravaha et al. [152] have highlighted the efficacy of a toothpaste containing pomegranate extracts as an additional treatment to complement routine periodontal therapies and have demonstrated that pomegranate flavonoids have an in vitro antibacterial property against the microorganisms responsible for gingivitis.

Nonetheless, there is still ample scope for research in this field.

2.8.6. Effects on Pregnant and Breast-Feeding Women

Recent studies demonstrated the importance of a nutritional supplementation with pomegranate. In particular, Al-Wazni et al. assessed the potential beneficial effects of three types of pomegranate extracts to fight urinary tract infections. Indeed, their findings demonstrated that the pomegranate peel extracts were able to inhibit *S. aureus* and *E. coli* bacteria, pathogens common to the urinary tract, in comparison to ciprofloxacin, used as reference molecule, and the negative control was represented by distilled water [153].

In 2017, Manouchehrian et al., through a clinical trial conducted on 80 healthy pregnan<sup>t</sup> women, demonstrated the potential effect of adding pomegranate paste to pregnan<sup>t</sup> women's diets on the incidence of neonatal jaundice. Indeed, the results revealed decreased values of bilirubin in the group fed with pomegranate paste with respect to control group [154].

A study carried out on pomegranate juice maternal supplementation, famous to be enriched with antioxidants and bioactive polyphenols, hypothesized the potential improvement of uterine and umbilical artery function with the consequent enhancement of fetal growth in the eNOS−/− mouse model of fetal growth restriction (FGR) without increasing fetal weight [155].

Similarly, Henning et al. envisaged the presence of one of the main pomegranate constituents, ellagic acid and its metabolites, in breast milk after the consumption of 8 ounces per day of pomegranate juice. As expected, they found detectable levels of ellagic acid and metabolites in breast milk after 14 days of nutritional supplementation. This could mean that the phenolic compounds delivering through breast milk could enhance infant health and development [156].

Needless to say, pomegranate represents an inexhaustible source of benefits, but for a safe use as therapeutic agen<sup>t</sup> on pregnan<sup>t</sup> and breast-feeding women other studies must be carried out.

### 2.8.7. Effects on Nervous System

Prior research considered neuroinflammation as the principal factor responsible for neurodegenerative diseases, including Alzheimer's and Parkinson diseases, as a consequence of neuronal degeneration after over-activating microglia in the brain [157].

Recent studies have promoted the neuroprotective activity of pomegranate due to the presence of several well-known anti-inflammatory components previously mentioned in the text (see Section 2.2).

In this context are well inserted the investigations carried out by DaSilva et al. in 2017. Their findings substantiated that urolithins, ellagitannin-gut microbial-derived metabolites present in PE, were able to lower important neuroinflammatory mediators such as NO, IL-6, PGE-2, and TNFα from lipopolysaccharide (LPS)-stimulated BV-2 microglia. Furthermore, they established the protection of SH-SY5Y and BV-2 (in vitro neuronal models) cell viability by hampering apoptosis and caspase 3/7 and 9 released from H2O2-induced oxidative stress [158].

Subsequently, Velagapudi et al. delved into urolithin mechanism of action by assessing not only the decrease of the previously mentioned mediators (IL-6, TNFα, NO) but underlining the relation between inhibition of neuroinflammation and Sirt-1 activation in BV2 microglia. Indeed, SIRT-1 levels, after treatment with 5 and 10 μM of urolithin, were much higher than control. This could sugges<sup>t</sup> that this compound should activate SIRT-1 in order to perform its neuroprotective activity e ffect [159].

More recently, Cásedas et al. confirmed these evidences toward another in vitro model, neuro-2a cells, by inducing oxidative stress. Data outlined that urolithin was able to improve mitochondrial activity (MTT assay), redox state (ROS formation, lipid peroxidation), and the activity of antioxidant enzymes (CAT: catalase, SOD: superoxide dismutase, GR: glutathione reductase, GPx: glutathione peroxidase). Furthermore, it raised the cytoprotective peroxiredoxin 1 and 3 expression and it was assessed to be a radical scavenger through ORAC (oxygen radical absorbance capacity) and DPPH assays [160].

Urolithin, contained in pomegranate juice, proved to also be e ffective in a rat model of Parkinson's disease induced by rotenone. Indeed, the nutritional supplementation with pomegranate juice enhanced postural stability, impeded oxidative damage and α-synuclein aggregation, permitted neuronal survival, improved mitochondrial aldehyde dehydrogenase activity, and preserved antiapoptotic Bcl-xL protein at the control level [161].

A recent clinical trial demonstrated the beneficial e ffects on memory derived from the daily consumption of pomegranate juice. In fact, for a period of 12 months, the diet of two hundred and sixty-one patients (aged 50–75 y) has been implemented with pomegranate juice (8 oz (236.5 mL) per day) or a placebo drink (8 oz, matched constituents of pomegranate juice except for pomegranate polyphenols). At six and 12 months, the memory measures (Brief Visuospatial Memory Test-Revised (BVMT-R) and Buschke Selective Reminding Test (SRT)) were carried out. From the results obtained, Siddarth et al. corroborated the hypothesis that the steady consumption of pomegranate juice may stabilize the ability to learn visual information over a 12-month period [162].

Taken together, reported data corroborate the concept that pomegranate may be considered a neuroprotector and developed as an agen<sup>t</sup> for treating neurodegenerative diseases.

#### **3. Toxicological Aspects of Pomegranate and Potential Interaction with Drugs**

Recently, some research has shown that pomegranate fruit can be considered part of a healthy diet and lifestyle without any risks or side e ffects [5]. Studies have shown that two doses of pomegranate extract (0.4 and 1.2 mg/kg body weight) produced no toxic reactions in rats in terms of food intake, weight changes, or behavioral or biochemical factors [163].

Heber et al. [164] carried out studies on 64 overweight subjects to assess the safety of the use of extracts in humans. Following the intake of 710 and 1420 mg capsules (containing 435 and 870 mg gallic acid equivalents, respectively), no adverse events occurred and no substantial di fferences in toxicity were estimated in any subject studied.

Other studies have been carried out on patients with carotid artery stenosis, showing that the intake of pomegranate juice (121 mg/<sup>L</sup> ellagic acid equivalent) for more than 3 years did not cause any toxic e ffect on blood parameters or liver, kidney, or heart functions [165].

Based on current research on pomegranate, considering its beneficial effects in cancer, cardiovascular diseases, etc., it was interesting to define the effects of pomegranate extracts on the cytochrome P450-3A, a liver enzyme system responsible for the metabolism of several drugs [7].

Studies in rats show that the administration of pomegranate juice possesses an inhibitory activity on the pharmacokinetics of carbamazepine, an anticonvulsant drug also substrate by the cytochrome P450-3A [7].

A single-dose randomized trial in healthy volunteers showed that treatment with pomegranate juice had no effect on the half-life or distribution time of intravenous administration of benzodiazepines with anxiolytic, hypnotic, anticonvulsant, and muscle relaxant properties, and no effect on maximum concentration or clearance after oral administration [7].

All of these findings strengthen the pomegranate value, making it an interesting and safe nutraceutical.
