*2.7. Antiviral Activities*

The hydrolysable tannins of pomegranate, including punicalin (13), punicalagin (9), gallic acid (18), and ellagic acid (8), have antiviral properties capable of modulating respiratory infections and influenza.

The antiviral properties of the polyphenolic extract of pomegranate are due to the inhibition of influenza virus RNA replication [117,123].

Similarly, phenols in the skin inactivate viruses through direct structural damage and indirect intercellular inhibition of viral replication. The virucidal e ffects of pomegranate phenolic compounds imply their interaction with the antigenic glycoprotein, hemagglutinin, present on the surface of some viruses (es. influenza viruses), which produce a loss of red blood cell agglutination [124].

In particular, Haidari et al. argued that punicalagin (9) is the e ffective component of pomegranate polyphenol extract able to block replication of the virus RNA, inhibit agglutination of chicken red blood cells (RBCs) by the virus of influenza H3N2, and have viricidal activity. They also investigated the potential synergistic e ffect of pomegranate polyphenol extract and oseltamivir (a well-known antiviral agent), outlining that oseltamivir in association with pomegranate polyphenol extract amplificated its anti-influenza power [125].

Sundararajan et al. in 2010 confirmed and implemented these data, strengthening that the direct anti-influenza activity of pomegranate polyphenols (PPs) is principally an outcome of PP-induced virion structural damage. Indeed, these components of pomegranates quickly block the influenza virus through a direct e ffect on the viral particle. They also suggested that this action might be separate from effects on hemagglutinin (HA) function. Moreover, they demonstrated the e fficacy of pomegranate PPs against H1N1 and H3N2 influenza viruses and against the reassortant H5N1 virus rg-VN/04 [126].

Furthermore, another study attributed to punicalagin the power to reduce the viral cytopathic effect on rhabdomyosarcoma cells, calculating an IC50 value of 15 μg/mL. This e ffect was also assessed in vivo, noticing a decrease of mortality in mice treated with a lethal dose of enterovirus 71 [126].

Reddy et al. focused on the potential e ffect against hepatitis C virus (HCV) of the ellagitannins extracted from pomegranate (*Punica granatum*) fruit peel. In particular, pure compounds punicalagin, punicalin, and ellagic acid exhibited in vitro the capability of blocking the HCV NS3/4A protease activity in a concentration-dependent manner with IC50 values of less than 0.1 mM (for punicalagin and punicalin), whereas IC50 for ellagic acid was achieved at 1.0 mM. These data were confirmed through in silico studies and observing a consistent reduction of HCV replication in cell culture systems. Data ex vivo pointed out the optimal bioavailability and the toxicity absence of these compounds [127].

In addition, a good activity of pomegranate extract toward adenovirus has been found. The IC50 and CC50 (50% cytotoxicity concentration) were estimated on HeLa cells with values of 165 ± 10.1 and 18.6 ± 6.7μg/mL, respectively, whereas the selectivity index (SI, calculated as the ratio of CC50 and IC50) on adenovirus amounted to 8.9 [128].

In their analysis, Houston et al. investigated the co-administration e ffects of pomegranate rind extract in conjunction with zinc (II) salts in order to challenge herpes simplex virus HSV-1 and its aciclovir-resistant. Data showed a potentiation factor by up to 5.5-fold. Regarding aciclovir-resistance, pomegranate rind extract exhibited an EC50 value amounting to 0.02 μg mL−1, whereas acyclovir displayed no activity [129].

Given the several reported studies that have proven the antiviral e ffects of pomegranate, Arunkumara and Rajarajanb surmised the possible inhibitory e ffect of *Punica granatum* against Herpes simplex virus-2 (HSV-2). In e ffect, their findings assessed that ethanolic peel extract suppresses HSV-2 at a concentration of 62.5 μg/mL. In order to understand the real responsibility of this action, the extract was subjected to bioactive compounds separation by bioassay-guided fractionation. Then, the single components were tested as antivirals. The key component for the activity seemed to be punicalagin, showing a total inhibition rate at 31.25 μg/mL. Moreover, bioactive compound analysis using the ADMET tool, established that human intestinal absorption (HIA) properties of acyclovir (ACY), gallic acid (GA), and ellagic acid (EA) had moderate adsorption values of 63.77, 53.69, and 61.39%, respectively, and punicalagin presented very strong plasma protein binding. In addition, docking studies highlighted that the most active component could interact with HSV-2 amino acids through several hydrogen bonds [130].

The global pandemic due to the spread of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has dramatically shaped queries on the necessity to find as soon as possible a cure for this disease. With this in mind, many e fforts, also with the aid of artificial intelligence, have been made to discover molecules able to interact with the viral proteins and cause the Sars-CoV-2 inhibition. Several compounds of natural origin were found to be able to do this. Among them, docking studies revealed that the ellagic acid, one of the already mentioned main components of pomegranate, has the potentiality to interact with important proteins involved in Sars-CoV-2 as RNA-dependent RNA polymerase (RdRp), angiotensin-converting enzyme 2 (ACE2), spike glycoprotein (SGp), and main protease (3CLpro) [131,132].

Furthermore, another study conducted on the viral main protease (3-chymotrypsin-like cysteine enzyme), held responsible for the COVID-19 control of duplication and life cycle management, assessed the potential of hydrolysable tannins (present also in pomegranate) as its natural inhibitors. Indeed, punicalin seems to establish H-bonds with the crucial catalytic residues of pocket spatial position [133].

From this research, it is deduced that pomegranate (especially peel extract) possess a prophylactic potential against viral epidemics and pandemics, specifically influenza. This may open up new avenues for research in the nutritional and medical science fields if the studies are made complete with in vivo experiments.
