*2.5. Antimicrobial Activities*

Antimicrobial agents are mainly applied on microorganisms that cause food poisoning (infected or toxin-producing agents) and on microorganisms that alter food by producing final metabolic products (catabolites) or enzymes with bad odor, unpleasant taste, problems of persistence, di fferent coloring, and/or health risk [106].

The antimicrobial e ffect of pomegranate and its products has been demonstrated in a large amount of research [107], in particular, polyphenols, flavonoids, and condensed and hydrolysable tannins derived from the fruit have been studied as promising agents to treat or prevent a broad range of infections [108].

The antimicrobial mechanisms of phenolic compounds engage the reaction of phenols with sulfhydryl groups of membrane proteins of the microbial cell, inducing a bactericidal e ffect due to membrane protein precipitation and inhibition of enzymes such as glycosyltransferase [109].

Food-borne disorders and urinary tract infections are treated typical in the Indian subcontinent through PoPx, while ellagitannins, punicalagin (9), ellagic acid (8), and gallic acid (18) in the pomegranate skin, as natural antimicrobial agents, have been extensively used against *Staphylococcus aureus* and *Escherichia coli* hemorrhages for their capability to precipitate membrane proteins and block enzymes such as glycosyltransferase, causing lysis. In vivo and in situ application of an 80% pomegranate skin (PoPx) methanol extract showed a growth reduction against *Listeria monocytogenes*, *Staphylococcus aureus*, *Escherichia coli*, and *Yersinia enterocolitica*. However, it has been reported that higher doses of PoPx (24.7 mg/mL) represent the lowest bactericidal concentration of *Listeria monocytogenes* [110].

Another study evidenced the powerful e ffect of *Punica granatum* L. peels' ethanolic extract (Tunisian Nana variety) against two Salmonella strains, *Salmonella Enteritidis* and *Salmonella Kentucky*, microorganisms resistant to the majority of antibiotics. Minimal inhibitory concentration (MIC) values ranged from 10.75 to 12.5 mg/mL regarding both strains. In particular, the inhibitory e ffect achieved by peel extract (using hydroethanolic and hydromethanethanolic mixtures) on *Salmonella Kentucky* achieved inhibition diameters of 22.2 and 22 mm, respectively [111].

Višnjevec et al. explored and evaluated the antimicrobial properties of ethanol and water extracts of pomegranate exocarp and mesocarp from Istria. The best antibacterial activity was recorded, here too, for ethanolic extract. Indeed, the microorganisms more susceptible to this extract were *C. albicans*, *C. parapsilosis, R. mucilaginosa, E. dermatitidis*, and *S. aureus*, with MIC ranging from 0.156 to 1.25 mg/mL. Instead, the best antimicrobial activities of the exocarp and mesocarp water extracts were found toward *S. aureus*, followed by *E. coli* without any antifungal activity [112].

Another use of pomegranate peel extract as antimicrobial agen<sup>t</sup> involved its participation in the synthesis of silver nanoparticles. The latter, due to the use of transmission electron microscopy (TEM) and scanning electron microscopy (SEM), demonstrated to be evenly distributed in the solution, with a spherical shape and size ranging from 20 to 40 nm and with an average particle size of 26.95 nm. Subsequently, tests evidenced the capability of nanoparticles to substantially inhibit Gram-negative and Gram-positive bacteria as *E. coli*, *P. aeruginosa*, *P. vulgaris, S. typhi*, *S. aureus, S. epidermidis*, and *K. pneumonia* already at 25 and 50 μg/mL [113].

As can be evinced from this section, pomegranate and, in particular, the peel extract, exhibited antimicrobial e ffects against a wide spectrum of bacteria. Unfortunately, in order to become e ffectively beneficial for the pharmaceutical field, much more has to be done regarding the deepening of the mechanism of action.

### *2.6. Prevention of Cardiovascular Diseases*

The main risk factors for the occurrence of coronary diseases is dyslipidemia, characterized by an excessive increase in low-density lipoproteins (LDL) and/or low levels of high-density lipoproteins (HDL) [114,115].

It is known that LDL oxidation contributes to atherosclerosis and the development of cardiovascular diseases [53,116].

Inhibition of LDL oxidation is considered a promising way to avoid the storage of foaming cells and, ultimately, cholesterol deposits in the arteries.

Due to its formidable antioxidant capacity, the extract obtained from the pomegranate skin has the power to suppress LDL oxidation and thus delay the advancement of atherosclerosis with an important decrease in foaming cell levels at the artery level.

The polyphenols present in pomegranate, punicalagin (9), gallic acid (18) and, to a lesser extent, ellagic acid (8), increase the expression and secretion of the liver enzyme paraoxonase 1 in a dose-dependent manner, thus reducing the risk of developing atherosclerosis [117].

Riaz and Khan in 2016 gave an insight into the anticoagulant, antiplatelet, and antianemic effects of *Punica granatum* juice by studying the hematological profile of the rabbits used as an in vivo model. Their findings evidenced a substantial increase in erythrocyte count, mean corpuscular hemoglobin concentration after 30 days of treatment, whereas red cell distribution width was significantly reduced. Instead, leucocyte count, hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and platelet count (PLT) were not modified at any dose. Furthermore, there was an important increase in bleeding time both after 30 and 60 days, and a significant rise in thrombin time (TT) and activated partial thromboplastin time ( αPTT) after 60 days, whereas prothrombin time did not change. Comparing the pomegranate ability in lowering cholesterol and the prolonged αPTT time, they speculated that the cholesterol reduction induced by *P. granatum* may lead to a decrease in the concentration of coagulation factors, influencing, in this way, αPTT. In conclusion, data illustrated that *P. granatum* significantly inhibited aggregation of platelets induced by adenosine diphosphate ADP and epinephrine (Epi) in the blood samples of animals treated already at 2 mL/kg for 30 and 60 days [118].

The hypothesis that pomegranate ameliorates the cardiac function has been also supported by Razani et al. Indeed, they administered 220 mL of pomegranate juice to one hundred hospitalized patients su ffering from unstable angina or myocardial infarction for 5 days in order, a the end, to evaluate cardiac markers and parameters. The results indicated a significant reduction in the intensity, occurrence, and duration of angina pectoris in patients with unstable angina. Moreover, the measurement of serum levels of troponin and malondialdehyde revealed a decrease of these factors. Nevertheless, other parameters like interleukin-6,tumor necrosis factor alpha, blood pressure, and heart rate were not a ffected [119].

More recently, the possible interaction between ethanolic peel extract of *Punica granatum* and doxorubicin (well known for its cardiotoxicity) was evaluated. The assessment was performed by measuring heart weight/body weight ratio, biochemical parameters, and histopathological changes. After the treatment with 100 mg/kg body weight, an increased heart weight/body weight ratio, in comparison with the doxorubicin-treated group, was observed as well as blood and tissue glutathione, superoxide dismutase, and catalase levels. On the contrary, creatine kinase, lactate dehydrogenase enzyme, and malondialdehyde levels appeared significantly reduced. Concerning the histopathological morphology, the only assumption was that doxorubicin modified myocardium through vacuolar alterations in the heart muscle and necrosis of heart muscle with remote cells increasing in the size in between the necrotic and fragmented muscle fibers. Instead, treatment with *P. granatum* peel extract displayed a protective e ffect with less histological changes such as irregular and spread vacuolation limited to subendocardial layers [120].

A recent clinical trial evidenced that the consumption of pomegranate juice improves the lipid profile and oxidative and inflammatory biomarkers of hemodialysis patients. The results outlined that triglycerides were decreased in the pomegranate juice condition and augmented in the control groups. On the other hand, high-density lipoprotein cholesterol was increased after PJ assumption, whereas total and low-density lipoprotein cholesterol were not a ffected. Systolic and diastolic blood pressure dramatically fell in the PJ condition, while total antioxidant capacity increased [121].

These data were also supported by Sohrab et al., who found in their study a decrement of systolic blood pressure and diastolic blood pressure; whereas, total cholesterol, low-density lipoprotein cholesterol decreased significantly compared to pre-trial values within the intervention group [122].

All these findings demonstrate the importance of pomegranate juice dietary supplementation in modulating cholesterol levels and preventing cardiovascular diseases.
