Pomegranate Health Properties

#### **Chapter 2**

## Antimicrobial Potential of Pomegranate Extracts

*Vildan Celiksoy and Charles M. Heard*

#### **Abstract**

The search for plant extracts with efficacious antimicrobial activity remains important, partly due to fears of the side effects associated with conventional antibiotics and to counter the emergence of resistant microorganisms. Pomegranate extracts have been used for millennia for their anti-infective properties, with activity more recently being attributed to its rich composition of ellagitannins and other secondary polyphenolic compounds. This chapter highlights the growing number of publications that have probed the activity of pomegranate extracts against microbes. Research generally supports folklore claims and has shown that pomegranate extracts possess unusual and potent broad-spectrum activities against Gram-positive and Gram-negative bacteria (planktonic and biofilm), fungi, viruses and parasites. Possible pathways/mechanisms of antimicrobial activity of pomegranate extracts are discussed and enhancement/potentiation of such activity using metal ions considered.

**Keywords:** antimicrobial, bacteria, fungi, viruses, parasites, polyphenols, pomegranate extracts, biofilm, tannins, punicalagin

#### **1. Introduction**

Infectious diseases caused by pathogenic microbes are a fundamental problem and remain one of the major factors behind high morbidity and mortality across the world, especially in developing countries. This is exacerbated by the worldwide emergence of antibiotic-resistant pathogens which has in turn given increased urgency to the discovery of new antimicrobial compounds, including those derived from plants [1, 2].

The pomegranate, fruit of the *Punica granatum* L. tree, is one of oldest recorded edible fruits and it has been used as a folklore medicine since ancient times. There are records of it being used to treat inflammatory diseases and disorders of the digestive tract in the Ayurvedic and Unani systems [3, 4]. In terms of infections, the ancient Egyptians used it in the treatment of tapeworms and other parasites [5], whereas other cultures have used pomegranates to treat diarrhea and dysentery [6–8], although at the time they would not have known that pathogenic microbes were responsible. In more recent times, the pomegranate has been extensively and scientifically studied for its antimicrobial potential in a diversity of areas such skin infections, dentistry, food preservation etc. [9].

The phytochemistry of pomegranate extracts is well described in the literature [10–12] and they are known to be rich in bioactive compounds especially

polyphenolics including anthocyanins and ellagitannins, in particular punicalagin, which is in the highest proportion [13]. As will be seen, it has become apparent that the pomegranate possesses unusual broad-spectrum potency against a wide range of species, which generally correlates with its polyphenol concentration.

In this chapter we aim to summarise published research into pomegranate extracts as antimicrobials and discuss some of the purported mechanisms behind such activity. Finally, the enhancement of antimicrobial activity by co-administration with metal ions is considered.

#### **2. Activity against bacteria**

*Staphyllococcus aureus* (*S. aureus)* and methicillin resistant *Staphyllococcus aureus* (MRSA) have received the greatest attention as targets for pomegranate extract activity. In 2010, the antibacterial activity of crude and purified extracts of pomegranate peel were assessed by Panichayupakaranant *et al.* 8 mg crude peel loaded discs showed 20 mm and 30 mm zone of inhibition against clinical isolates of *S. aureus* and *E. coli*, respectively. The purified peel extract discs, loaded up to 8 mg, exerted a range of zones of inhibition between 15-20 mm for *S. aureus* and 20-30 mm for *E. coli*. Using standardized peel extract, minimum inhibitory concentrations (MIC) values of 0.016, 0.008, and 0.008–0.016 mg/mL were obtained for *S. aureus*, *S. epidermidis* and *Propionibacterium acnes* respectively. Tetracycline was used as a positive control in this study and standardized pomegranate rind extract showed lower activity in zone of inhibition assays, with tetracycline also showing a lower minimum inhibitory concentration (MIC) [14]. A methanolic extract of pomegranate peel inhibited biofilm formation and eradicated pre-formed biofilm of *S. aureus*, MRSA, *E. coli* in the concentration range 25 to 150 μg/mL [15]. In the same study, ellagic acid showed biofilm inhibition and eradication activity at somewhat lower concentrations (5–40 μg/mL) than pomegranate peel extract. Furthermore, while pomegranate extract was able to inhibit the growth of *S. aureus*, it also suppressed enterotoxin production [5].

Pomegranate extracts have shown antimicrobial activity against to a range of oral microbes. It has been found that pomegranate extract powder at 1 mg/mL was effective against primary and secondary colonizer bacteria of dental plaque: *F. nucleatum, P. gingivalis, P. intermedia, S. mutans* and *A. actinomycetomomitans* [16]. In another *in vitro* study, pomegranate alcoholic extracts have been tested on bacteria which are collected from patients who have tooth decay or periodontitis and inhibited a range of bacteria in both planktonic and biofilm conditions [17]. Synergistic bactericidal activity against *S. mutans* and *R. dentocariosa* was reported for pomegranate extract in combination with other plant polyphenolic extracts, honey and myrtle [18].

Moreover, 'standardized' pomegranate peel extract showed higher antimicrobial activity than other parts of pomegranate (flower, leaf, stem) and ciprofloxacin (2 mg/mL) against *S. mutans, Salmonella mitis* and *L. acidophilusin* in a zone of inhibition assay [19]. Again, pomegranate gel showed an inhibitory activity against *S. mutans, Salmonella sanguis,* and *S. mitis* [20]. This gel also showed antiadhesive activity against *S. mutans* and *S. mitis* at lower than minimum inhibitory concentrations to a glass surface. In addition to inhibition activity on bacterial growth and biofilm, pomegranate extracts showed antiadhesive activity for *S. mutans* adherence on tooth surface in orthodontic treated patients [21]. In other clinical studies, the antiplaque effect and prophylactic benefits of pomegranate have been highlighted [22]. Recently, a systematic review and meta-analysis has been carried out by Martins *et al.* [23], where natural antimicrobial phenolic compounds were


#### *Antimicrobial Potential of Pomegranate Extracts DOI: http://dx.doi.org/10.5772/intechopen.95796*


#### **Table 1.**

*Antibacterial activity of pomegranate extracts against different bacteria.*

compared with synthetic antimicrobials by using 16 clinical studies for qualitative analysis, and 12 studies for meta-analysis. For the meta-analysis, six clinical trials were evaluated for the comparison of natural antimicrobial phenolic compounds, including pomegranate extract mouthwash, and synthetic antimicrobials. It was found that natural antimicrobial phenolic compounds are less effective than chlorhexidine for biofilm control, although it showed similar reduction of the oral microbes count which was sub-grouped as total microorganisms, *Streptococcus mutans*, and *Streptococcus spp.* according to type of microorganisms.

Due to its antimicrobial and antioxidant properties, pomegranate extract has been studied for its preservation potential use in the food industry. Kannat *et al.* [24] did a study to evaluate the antimicrobial activity of pomegranate peel against common food spoilers and potential pathogens. It was shown that pomegranate peel extract increased the shelf-life of chicken and meat products and showed antimicrobial activity against to *S. aureus, B. cereus* with a minimum inhibitory concentration at 0.01%. However, it did not show antimicrobial activity for *E. coli* and *S. typhimurium* even at higher concentrations. Other researchers showed that pomegranate extracts were less effective against Gramnegative compared to Gram-positive bacteria, probably due to the differences in cell wall structure [25, 26]. Moreover, pomegranate has been studied in a novel and smart multi-functional hydrogel (MFH) system as a food packaging material since it is easy to monitor of the color change due to changes in conditions such as pH and temperature. The MFH with pomegranate extract showed promising antimicrobial activity on pasteurized milk and cheese over a 7-day period [27]. Pomegranate peel extract was also added in a film formulation to produce a material for food packaging materials with antimicrobial and antioxidant effects. This film formulation was found to restrict the growth of *L. monocytogenes* in pork samples inoculated with this bacterium [28].

The activity of pomegranate extract against bacteria is summarized in **Table 1**.

#### **3. Activity against fungi**

Treatment of fungal infections is a big challenge because of the eukaryotic nature of fungal cells that have similarity with host cells. While there are some drugs in the treatment of fungal infections available in the clinic, they are limited and there is a need for new alternatives [29, 30]. There are reports showing antifungal activity of pomegranate extracts, especially against *Candida* species [5, 7, 31], which are part of the normal microbiota of human gastrointestinal, oral, and vaginal mucosae. However, they can cause superficial infections and especially in immunosuppressed patients, they can cause severe infectious problems. In one study, punicalagin showed superior antifungal activity than the conventional fluconazole in an *in vitro* time-kill assay. In addition, punicalagin caused a significant change in *Candida* morphology, and alteration in budding pattern and pseudo hyphae when yeasts were treated with a sub-inhibitory concentration of punicalagin [32]. In another study, pomegranate

*Antimicrobial Potential of Pomegranate Extracts DOI: http://dx.doi.org/10.5772/intechopen.95796*

extract showed superior inhibitory action against *C. tropicalis* while fluconazole and voriconazole, which are commonly prescribed azoles for fungal infections, have been ineffective against *C. tropicalis* [33].

The potential of pomegranate extract has been studied against fungi in *in vitro* biofilm assays. Microbes in biofilms have substantially different characteristics to those of their free-living planktonic counterparts [34, 35]. In particular, microbes in biofilms are concealed and therefore protected from antifungal agents and plant extracts [36, 37]. Pomegranate extract and its one of the major components ellagic acid were shown to exert a reduction in biofilm formation and eradicated pre-formed biofilm of *C. albicans* in an *in vitro* biofilm study [15]. Spray-dried microparticles containing pomegranate extract showed antifungal activity in *in vitro* assays under both planktonic and biofilm conditions [38]. In addition, the inhibitory effect of pomegranate extract on the growth of *Candida albicans* was demonstrated in an *in vivo* study [31]. In another study, similar effects have been obtained against to *Candida mycoderma* using different parts of pomegranate, fresh fruit and sterile juice [39].

In addition to *Candida* species, pomegranate extracts showed inhibitory activity against dermatophytes, which are fungi which use keratin as a source of nutrition and may cause infection in keratinized tissue parts such as nails, skin and hair follicles. Pomegranate peel extract and punicalagin exerted potent antifungal activity against to *T. rubrum* (125 μg/mL), *T. mentagrophytes* (125 μg/mL), *M. canis* (250 μg/ mL) and *M. gypseum* (250 μg/mL). Punicalagin at a concentration of 62.5 μg/mL also inhibited *T. rubrum* spore germination, and it was further found that punicalagin 62.5 μg/mL and nystatin 0.78 μg/mL showed similar inhibition in hyphal growth of *T. rubrum* [40]. Moreover, pomegranate extracts have been researched for use as natural preservatives due to their antifungal (in addition to antibacterial) activities [41]. Pomegranate peel extract showed inhibition activity against to *Aspergillus* 


**Table 2.**

*Antifungal activity of pomegranate extracts against different fungi.*

*niger* and *Aspergillus parasiticus* in a zone of inhibition assays [42]. Pomegranate extract showed inhibitory effects against fungal pathogens which are responsible for fruit and vegetable decay. Punicalagin was proposed as the main compound in the extracts providing the observed antifungal activity and it has been found effective in mycelial growth inhibition against phytopathogenic filamentous fungi such as *Fusarium vertillicoides, Mucor indicus, Penicillium citrinum, Rhizopus oryzae*  and *Trichoderma recei* [43]. Also, the growth rate of pathogens presented a negative correlation with total punicalagin content, and it has thus been suggested that pure punicalagin may be used as a control agent in storage disease to prevent the excessive use of synthetic fungicides [44, 45].

The activity of pomegranate extract against fungal microbes is summarized in **Table 2**.

#### **4. Activity against viruses**

Pomegranate extracts have been examined as an alternative treatment for viral infections [46–48]. A number of studies have shown that polyphenolic compounds have broad-spectrum antiviral activity, by inhibiting viral DNA and RNA, and directly binding the viral particles. It has also been suggested that polyphenols could provide antiviral activity during intracellular replication [49–52].

Pomegranate peel extract showed antiviral activity against the influenza virus. In a study by Sundararajan *et al.* [53], complete inactivation of influenza virus was observed with 1600 μg/mL pomegranate polyphenols, and 400 μg/mL of same extract showed 99% or more titer reduction in only 5 minutes treatment. This result was similar to another study which showed complete inactivation of H3N2 influenza virus within 30 minutes of treatment and a significant viral reduction with approximately 1 μg/mL pomegranate polyphenols. An *in vitro* study, showed that pomegranate polyphenol extract inhibited viral replication in addition to its virucidal effect – they also obtained same activity for punicalagin and suggested punicalagin is the main compound in pomegranate extract for antiviral activity [54]. In an *in vivo* mouse model study, pomegranate polyphenols applied to the lung were found to reduce influenza infection, without toxic effect to the host [55, 56].

*Hepatitis C* virus (HCV) is the main factor in end-stage liver disease and approximately 170 million people are chronically infected with HCV. Pomegranate ellagitannins, punicalagin, punicalin and ellagic acid, blocked and inhibited the NS3/4A protease which is a viral polyprotein responsible for processing and replication in HCV. Moreover, punicalagin and punicalin significantly decreased the HCV replication in an *in vitro* cell culture system [57]. The more prevalent adenovirus (ADV) in Hep-2 host cells has also shown susceptibility to pomegranate crude extract, fractions, and main phenolic compounds. It has been found that a n-butanol fraction of pomegranate peel extract and gallic acid showed the highest antiviral activity against ADV. Furthermore, the crude extract, n-butanol fraction and gallic acid inhibited ADV replication in the post-adsorption phase [58].

*Herpes simplex* virus (HSV) is from the *Herpes* viridae family and infects a high proportion of the populous. HSV-1 is generally responsible for cold sores and encephalitis, whereas HSV-2 is the main causative agent of anogenital infections, which can also infect neonates via the mother [59, 60]. Pomegranate rind extract (PRE) and its major ellagitannin compound, punicalagin, showed virucidal activity against HSV-1. While punicalagin has greater virucidal activity than an equivalent mass of pomegranate rind extract, PRE showed better antiviral activity than punicalagin. Moreover, PRE demonstrated comparable activity to acyclovir against HSV-1 and

*Antimicrobial Potential of Pomegranate Extracts DOI: http://dx.doi.org/10.5772/intechopen.95796*


**Table 3.**

*Antiviral activity of pomegranate extracts against different viruses.*

HSV-2, in addition to antiviral activity against acyclovir-resistant HSV-1 [48]. PRE is thus a promising new alternative treatment for HSV-1 since currently acyclovir is the gold standard treatment in HSV infections [61].

Studies have suggested that the antiviral activity of pomegranate extract originates from its hydrolysable tannins and polyphenols, especially punicalagin and gallagic acid. However, in one study, four flavonoids, ellagic acid, caffeic acid, luteolin and punicalagin, from pomegranate peel extract were studied against influenza virus and only punicalagin showed an inhibitory effect. The antiviral activity of pomegranate rind extract has been patented in Japan based on pomegranate peel extract ability to prevent the growth and kill viruses on the surfaces [46, 47]. The activity of pomegranate extract against viruses is summarized in **Table 3**.

#### **5. Activity against parasites**

Parasitic infections remain a significant global problem, affecting the health of hundreds of millions of people annually, especially in countries with low economic and social conditions. In addition, the increased world-wide resistance to conventional drugs is making most of currently used drugs less effective. As a result of this situation, the development of new drugs from medicinal plants for parasites is as important as for other microbes [62]. Different parts of *Punica granatum* L., root, stem bark, and rind of fruit, have been used commonly as vermifugal and taenicide agents [63]. The antiprotozoal activity of the pomegranate has been determined and in folkloric medicine, it has been used as anthelminthic especially against tapeworms and for diarrhea [64, 65]. A methanolic extract of pomegranate leaves showed nematicide activity and hepatoprotective activity against carbon tetrachloride induced hepatoxicity [66]. Extracts of pomegranate showed anti-schistosomal activity against *Shistosoma mansoni* in both *in vitro* and *in vivo* conditions [67].


#### **Table 4.**

*Antiparasitic activity of pomegranate extracts against different parasites.*

In addition, it caused reduction or complete loss of motor activity, lethality and ultra-morphological changes in adult worms [68]. There is thus potential for the treatment of schistosomiasis.

Al-Musayeib *et al.* reported the antiparasitic activity of pomegranate rind extract against *Plasmodium falciparum* [69]. Pomegranate juice was found to exert dose-dependent activity against *Leishmania major* promastigotes and, at >80 μL/mL, gave significantly greater reduction than the positive control, Pentostam. Furthermore, mice that were orally treated with pomegranate juice, showed significantly reduced cutaneous leishmaniasis lesions compared to untreated mice [70]. Calzada *et al*. demonstrated pomegranate antiprotozoal activity against *Entameoba histolytica* and *Giardia lamblia* that cause diarrheic dysentery [71]. Pomegranate peel suspension also affected *C. parvum* in different stages and finally caused parasite death in an *in vivo* murine model; furthermore, pomegranate suspension did not cause any negative change in the mice ileal tissue [72]. In another study, pomegranate extract showed activity against *T. vaginalis*, both *in vitro* and clinically. Patients with *T. vaginalis* infection were treated with pomegranate juice and symptoms were found to have cleared after two months [73]. The activity of pomegranate extract against parasites is summarized in **Table 4**.

#### **6. Potential mechanisms of antimicrobial activity of pomegranate extracts**

From the preceding sections it is clear that there is compelling evidence demonstrating the broad-spectrum antimicrobial activity of pomegranate extracts [74–76]. However, the precise mechanism behind this activity is not fully

#### *Antimicrobial Potential of Pomegranate Extracts DOI: http://dx.doi.org/10.5772/intechopen.95796*

understood. The mode of antimicrobial action of polyphenols, in general, is also unknown, although some suggested mechanisms include membrane disruption, toxicity against microorganisms, the ability of complex formation with metal ions and enzyme inactivation [77–79]. The antimicrobial activity of pomegranate has been associated with polyphenolic tannins, especially punicalagin and ellagic acid content in the extract [80–82]. However, pomegranate extracts are a complex mixture containing a variety of secondary compounds and interplay between these components may be a factor in antimicrobial activity, with multiple mechanisms operating independently [83].

An antimicrobial mechanism suggested for polyphenolic compounds is based on the precipitation ability of these compounds with bacterial cell membrane proteins which leads to bacterial cell lysis [84]. In addition, polyphenols could inhibit microbial enzymes by reacting with sulfhydryl groups or nonspecific interactions with proteins [85]. In that vein, phenolic compounds can bind the protein sulfhydryl groups and make them unavailable for microbial growth [86]. In addition, it has been reported that polyphenols can damage the microbe respiratory chain by decreasing the oxygen consumption and thus limiting the oxidation of NADH [87].

It has been hypothesized that the antibacterial activity of phenolic acids and flavonoids could cause a decrease in membrane fluidity by giving damage to the bacterial cytoplasmic membrane [88]. Phenolic acids can cause hyper acidification when they interphase with the plasma membrane. This situation would cause an alteration in cell membrane by making it more permeable. This mechanism could explain why phenolic acids show different antimicrobial activity levels against different pathogenic microorganisms [89, 90]. One of the possible mechanisms could be related to hydroxyl groups of polyphenols. The position of OH group in the aromatic ring and the length of saturated side chain could be a cause of antimicrobial activity of polyphenols [91]. Hydroxyl groups can bind to bacteria cell membranes and interfere with processes, such as ion pumping. In addition, OH groups can interact with active site of enzymes and disturb the metabolism of microorganisms [91].

Pomegranate extract exerted an inhibition activity against biofilms, in addition to their planktonic counterparts. Since microbes act differently under biofilm conditions compared to their planktonic form, there are some suggested pathways about polyphenols biofilm eradication and formation inhibition activities, although still unconfirmed. The mechanism behind growth and biofilm inhibition by pomegranate extracts cause protein precipitation and enzyme inactivation [81, 92]. Pomegranate extract could precipitate proteins which play major role in biofilm formation, like adhesins. Moreover, major hydrolysable tannins in pomegranate extract such as ellagic acid can change the surface charge and reduce the cell-substratum interactions and biofilm formation and development on different surfaces [93]. It is well known that tannins have astringency properties, and this feature can play a part in biofilm disruption [94, 95]. Different studies have shown the activity of pomegranate on bacterial attachment and therefore biofilm formation. It has been demonstrated that *Punica granatum* L. showed a specific antimicrobial action on dental plaque, which is a complex biofilm on tooth, by inhibiting adherence mechanism of oral microbes to dental surface via disturbing polyglucan synthesis [17, 96, 97]. Moreover, Vasconcelos *et al*. [98] used *Punica granatum* L. in a gel formulation using increasing and doubled concentrations of the diluted solutions of the gel with ranging concentrations from 1:1 to 1:1024, and similar results obtained. The gel formulation inhibited the adherence of different bacterial strains and a yeast, *C. albicans*, in the oral cavity and affected preformed biofilm.

There are some reports suggesting that the inhibition of quorum sensing (QS) could play role in the biofilm inhibition activity of pomegranate [99, 100]. QS is a communication system between bacteria in a biofilm, and provides a network

involving nutrients, defense against other microorganisms, virulence and biofilm formation. More importantly, QS helps microbes to escape from host immune response [101, 102]. Therefore, inhibition of QS is quite important in order to overcome microbial infectious diseases and resistant pathogenic microbes. For the evaluation of QS inhibitors, *Chromobacterium violaceum* has been used as a biosensor since it produces violacein, purple pigment color, in response to QS regulation [103]. Pomegranate inhibited the QS of two bacterial strains which are *Chromobacterium violaceum* (by affecting purple pigment production) and *P. aeruginosa* (by decreasing bacterial swarming motility) [104, 105]. In another study, different compounds from herbs, fruits and plant extracts have been studied for their QS activity, with resveratrol and pomegranate extract demonstrating the highest inhibition activities. The QS activity of pomegranate has been associated with ellagic acid content of pomegranate extract (85% punicalagin, 7% free ellagic acid) since ellagic acid showed 86% inhibition at a low concentration of 4 μg/mL. However, the anti-QS activity of punicalagin is also believed important in pomegranate extracts [106]. Tannin-rich fraction of pomegranate rind extract showed inhibition of biofilm formation and motility of *E. coli* and repressed the expressions of curli genes (*csgB* and *csgD*) and various motility genes (*fimA, fimH, flhD, motB, qseB*, and *qseC*) [107]. Similarly, punicalagin significantly decreased the expression of QS-related genes (*sdiA* and *srgE*) of Salmonella [108].

The chemical structure of tannins has importance in bacterial growth inhibition. For example, hydrolysable tannins were found to give lower minimum inhibitory concentration than condensed tannins [109]. The degree of galloylation has an effect on antibacterial activity since a higher degree of galloylation have more protein binding capacity and higher affinity for iron. However, the antibacterial activity is not only correlated to galloyl groups and galloylation, also it is correlated to configuration of the digalloyl or trigalloyl groups that attached to glucose core [110–112]. In addition, free galloyl groups have a major role in antimicrobial activity of ellagitannins which are abundant secondary compounds in pomegranate extracts [12, 113]. Punicalagin showed the broad-spectrum antimicrobial activity and it has a gallagyl moiety [114]. However another ellgitannin, granatin A, which does not have free galloyl groups, did not show antibacterial activity [115]. In a study done by Reddy *et al.*, ellagic acid, gallagic acid, punicalin and punicalagin were purified and tested for their antiplasmodial and antimicrobial activities. Gallagic acid and punicalagin showed the strongest effects on the growth

**Figure 1.** *Reduction of punicalagin, punicalin and HHDP to ellagic acid, adopted from Seeram* et al. *[3, 12].*

#### *Antimicrobial Potential of Pomegranate Extracts DOI: http://dx.doi.org/10.5772/intechopen.95796*

of bacteria and fungi and it has been suggested that the ellagic acid moiety is not important compared to the gallagyl and hexahydroxydiphenol (HHDP) moieties for the inhibition of microbes [116]. The degradation of punicalagin to ellagic acid, via punicalin and hexahydroxydiphenic acid is shown in **Figure 1**.

The antimicrobial activity of plants has been studied extensively and many active secondary compounds have been identified. However, it should not be ignored that plant extracts with antimicrobial activities contain potentially many secondary compounds. Therefore, it is not easy to attribute the biological activity of plant extracts to only a single compound or a group of secondary compounds. There is a high possibility that plant extracts show antimicrobial activity due to synergistic effect of different compounds [117].

#### **7. Enhanced antimicrobial activity of pomegranate extracts with metal ions**

There are many reports showing the antimicrobial activity of heavy metals such as iron, copper, silver, manganese and zinc, while many bacteria have mechanism for the detoxification of heavy metals [118, 119]. However, although metal ions have a strong antimicrobial effect, they can also be cytotoxic to human cells, therefore, the use of these metals may have limitations in healthcare [120, 121].

Stewart *et al*. [122] investigated the potentiated antimicrobial activity of pomegranate rind extract (PRE) in combination with metal ions. In their study, the aim was to exert short term exposure of pomegranate rind extract and ferrous sulfate combination on bacteriophage levels for 3 minutes. This combination showed highly significant synergistic activity and reduced the bacteriophage levels in a short-term exposure. This short screening time was necessary for this experiment due to low stability of pomegranate rind extract/ferrous salt solution which, via a Fenton reaction caused Fe2+ to oxidize to Fe3+ with concomitant solution blackening. To overcome this instability problem, potentiated/synergised antimicrobial activity of pomegranate rind extract has since been examined using alternative metal ions [48, 123, 124].

McCarell *et al.* [123] investigated the antimicrobial activity of PRE with 4.6 mM FeSO4, CuSO4, MnSO4, ZnO and also studied antimicrobial activity of PRE/metal salt combinations plus vitamin C which was added as a stabilizer. They observed significant synergistic antibacterial activity against *E. coli, Ps. Aeruginosa, S. aureus* and *P. mirabilis* when they combined PRE with Cu (II) ions. Moreover, with the addition of vitamin C as antioxidant, the antimicrobial activity increased significantly for PRE/Fe (II) and PRE/Cu (II) combinations against *S. aureus*. In another study, researchers used the vanillin complexes with different metal ions using the agar diffusion method and it was found that the vanillin and metal salts showed an enhanced activity against *S. aureus, E. coli, K. pneumanie, P. aeruginosa and C. albicans*. The results from both studies indicated that the addition of metal ions, especially copper salts, can significantly enhance antibacterial activity of a natural product [123, 125].

Significantly enhanced virucidal activity of PRE was later observed against HSV-1, HSV-2 and acyclovir-resistant HSV-1 by Houston *et al.* [48] in combination with different Zn (II) ion salts, including zinc sulphate, zinc citrate, zinc stearate and zinc gluconate, with a maximum of 6 log reduction observed. Unlike PRE and Fe2+, this activity was not time-limited, and was not associated with blackening. Importantly, this activity was also retained when applied to epithelial surfaces prone to *Herpes* infection, including buccal and vaginal mucosae [126], indicating potential treatment for cold sores and anogenital *Herpes*.

The mechanism for the synergistic antimicrobial activity of pomegranate extract in combination with metal ions is not clear, although there are some putative suggested mechanisms for this enhanced antimicrobial activity. For instance, it has been suggested that pomegranate tannins can form a 'complex' with metallic ions and this complex could show enhanced toxicity to microbes [127]. Furthermore, PRE could show enhanced activity due to redox cycling of the associated metal ion which increases local levels of reactive oxygen species (ROS). For example some antibiotics e.g. bleomycin showed enhanced ROS production via the ability to bind to ferrous ions which resulted in enhanced toxicity against microbes [128].

The enhancement of antimicrobial activity of pomegranate rind extract with metal ions is important in terms of improved efficacy against antibiotic resistant pathogens, since this enhancement could reduce resistance of microbes by inhibiting their microbial adaptability features [8, 32].

#### **8. Conclusions**

The pomegranate has a long history of use as a folklore medicine for its ability to address microbial infections. Published research, as outlined in this chapter, clearly supports this and has shown that pomegranate extracts possess an unusual and potent broad-spectrum of activities against bacteria, fungi, viruses and parasites.

There is some variation in the literature in terms of the levels of antimicrobial activity of pomegranate extracts, which could be attributed to different harvesting time and type of pomegranate cultivars, and use of varying microbial strains. However, it is also apparent that different workers have used a range of approaches to obtain 'pomegranate extract', with extraction methods sometimes being poorly described. As such, a lack of standardized test extracts presents a challenge in attempting to make quantitative comparisons. As a complex mixture, pomegranates extracts have the innate ability to inhibit resistance, even more so when used alongside a synergizing metal ion.

#### **Acknowledgements**

We would like to thank to Turkish Ministry of Education for supporting Vildan Celiksoy's PhD project.

#### **Conflict of interest**

The authors declare no conflict of interest.

*Antimicrobial Potential of Pomegranate Extracts DOI: http://dx.doi.org/10.5772/intechopen.95796*

### **Author details**

Vildan Celiksoy and Charles M. Heard\* School of Pharmacy and Pharmaceutical Sciences, Cardiff University, United Kingdom

\*Address all correspondence to: heard@cardiff.ac.uk

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 3**

## Role of Pomegranate in the Management of Cancer

*Amulya Thotambailu, Deepu Cheriamane, Manjula Santhepete, Satheesh Kumar Bhandary, Jiju Avanippully and Prakash Bhadravathi*

#### **Abstract**

Pomegranate (*Punica granatum*) has been used since ages as a folk medicine. Studies have shown that pomegranate extracts have a role in various signaling pathways involved in inflammation, cellular transformation, hyperproliferation, angiogenesis, initiation of tumorigenesis, and eventually suppressing the final steps of tumorigenesis and metastasis. In this chapter, we have discussed some of the polyphenolic constituents present in pomegranate and their medical value, and we then discussed studies on chemopreventive/chemotherapeutic properties of pomegranate against various types of cancer, such as skin, prostate, colon, head and neck and lung cancers in cell culture systems, animal models and humans.

**Keywords:** Pomegranate, cancer, antioxidants

#### **1. Introduction**

The Pomegranate fruit (*Punica granatum* L.) is a perineal fruit of the family punicaceae, the fruit comprises of white to deep purple seeds covered in a spongy membrane which is further covered by the pericarp. It is available across the globe as a well-established folklore medicine owing to its anti-oxidant and antiinflammatory properties. Various useful medicinally active components are present in the peel, seed, flower and even the leaves. The phenolic components comprise the major medicinally active part of the pomegranate extract along with minerals like magnesium, phosphorous, sodium and potassium [1–3]. There are four groups of phenolic compounds present in pomegranate namely the groups with anthocyanin pigments, hydrolysable tannins like punicalagin, ellagic acid and hydrolysable tannins. All of which contribute to the antioxidant activity. Moreover, it is rich in flavonoids and tannic acids which further adds to its the medicinal value. The antioxidant and anti-inflammatory potential of pomegranate juice and pomegranate extracts puts it apart from other fruits owing to the high concentrations of hydrolysable tannins and anthocyanins along with the polyphenols [4]. Among the various parts of the fruit, the peel and lamella which are the main on edible parts of the fruit has majority of the phenolic contents compared to the other edible parts. The peel accounts for more than half of the total antioxidative potential of the fruit and their anti-proliferative activity [5, 6]. The seed coat of the fruit also presents with numerous organic acids including citric acid and ascorbic acid [7]. Among the major biomedical advantages of pomegranate is the anti-cancer activity since pomegranate and its various components has been prove to assist the treatment of cancer and show immunomodulatory activity [8]. This chapter will be discussing on the anti-cancer activity of pomegranate and its antioxidant activity.

Cancer is one of the most common disease conditions which is becoming the leading cause of death even when detected in its early stages. In the year of 2021, almost 2 million new cancer cases are expected to happen just in the united states. The cancer death is reducing with each decade comparing to the initial few decades since its peak. With each passing year, there is rapid improvement in the cancer treatment strategies [9]. Pomegranate components can be used for treatment of many ailments as such or as an adjuvant in the treatment. One of the common problems related to cancer therapy is the lack of specificity in differentiating the cancer cells from the normal cells which manifests problems in the oral cavities as mucositis or candidiasis. This shows the prospect of using pomegranate extracts as an adjuvant in normal cancer chemotherapy in order to improve the quality of life of the people undergoing treatment. Also, the rind extract rich in the tannin punicalagin when used in combination with zinc is shows healing activity in the oral cavity due to the anti-inflammatory activity [10, 11].

#### **2. Pomegranate and prostate cancer**

Prostate cancer is the most prevalent type of cancer in men with an incidence rate above 30 worldwide [12]. From the multicenter studies conducted in human prostate cancer using pomegranate extracts rich in polyphenols, the extracts were found to cause an inhibition in the proliferation of the cells in both in *vivo* and *in vitro* studies. Hence the study demonstrated a significant anticancer activity through the inhibition of invasion and proliferation of the cancer cells [13]. The initial stages of prostate cancer will be testosterone dependent and this can be treated with normal radiation or chemotherapy but not in the late stages which shows no dependence with testosterone. The Further studies on the extracts from different parts of pomegranate showed a synergic activity in the anti-proliferative activity of other components of pomegranate. The seed oil from pomegranate acts as a synergistic when used in conjunction with the juice rich in polyphenols in the prevention of proliferation even though it does not have any anti-proliferative effect alone [14].

The studies on LNCaP cell lines which are modified to over express androgen receptors so that a situation similar to that of androgen independent prostate cancer. By using the different pomegranate extracts rich in polyphenols, the study later on showed a decrease in expression of the gene for the androgen synthesizing enzymes. Since the down regulation of androgen receptors is evident from the study, pomegranate extracts can be of use in the treatment of prostate cancer with an up-regulation of androgen receptors [15]. In androgen independent prostate cancer, there is an observed activation of the nuclear factor NF-κB. The activation of this nuclear factor is a common event in many types of cancer including breast cancer and cervical cancer [16]. In the molecular studies conducted using pomegranate extracts on the activity of NF-κB. It was found that the pomegranate extracts were able inhibit the NF-κB activity which was shown in the androgen independent cells, DU145 with increasing doses. Congruent results were obtained from the electro mobility shift assay conducted on the same cells using pomegranate extracts. In the DU145 and CL-1 cells which are the androgen independent cell lines, the activity of NF-κB was found to be activated through the TNF-α. Pomegranate extracts showed

*Role of Pomegranate in the Management of Cancer DOI: http://dx.doi.org/10.5772/intechopen.97188*

promising activity in the inhibition of NF-κB cells activated in this way as well. In the LAPC4 xenograft induced model of cancer, the extracts from pomegranate was found to delay the initiation of prostate cancer through prevention of proliferation of the cells [17].

Punicalagin is an important polyphenol constituent of pomegranate and as discussed before, the antioxidant activity of which is very evident in the cancer cells. The antiproliferative activity of punicalagin was examined in previous studies using the DPPH assay and the lipid peroxidation inhibition assays. Along with this, the study checked the cytotoxic activity and viability effects were also determined using punicalagin. It was found that punicalagin inhibited proliferation of cancer cells in prostate cancer and that the prostate cancer cells remained intact in the presence of punicalagin which was further supported by evidences from cell viability assays. The antioxidant activity of the polyphenol was further shown in the DPPH free radical scavenging assay which showed that it scavenged the free radicals in a dose dependent manner. The lipid peroxidation was also inhibited in the presence of punicalagin. PC-3 is another major cell line which is involved in prostate cancer and the polyphenol was found to reduce the PC-3 cells through apoptosis with higher concentrations [18, 19].

Further, it was found that pomegranate extracts affect the bio synthesis of androgens from the studies conducted using prostate cancer models. In the in vivo study conducted on the animal model using PTEN (Phosphatase and tensin homolog) knockout mouse which represents prostate cancer, there was observable reduction in the levels of steroids in the serum and in the case of in vitro studies using prostate cancer cell lines LNCaP and 22RV1, pomegranate extracts were found to cause a fall in the production of androgens. The in vitro and in vivo date obtained from various studies further shows the possible activity of pomegranate extracts in the treatment of Prostate cancer [20].

#### **3. Pomegranate and breast cancer**

Breast cancer is the most common type of cancer diagnosed in women and the leading cause of death due to cancer in women with over 2 million cases being diagnosed from recent studies [21]. The major causative factor for the cancer proliferation in breast cancer proliferation is estrogen and the enzymes which catalyzes the production of estrogen. The enzyme aromatase aids in the conversion of androgen into estrogen. So, the inhibition of this enzyme can further aid in the treatment of breast cancer. In vitro studies conducted on one of the major constituents of the pomegranate namely extract ellagic acid and urolithins A and B showed promising results on the inhibition of aromatase enzyme. The placental microsome aromatase assay conducted on ellagitannin derived compounds from pomegranate extracts namely, methylated urolithin B, methylated urolithin A and urolithin A further showed the aromatase inhibiting activity of pomegranate extracts. Which in turn inhibits the proliferation of cancer cells [22].

From the in vivo studies conducted on mammary organ culture in mice using the pomegranate seed oil rich in punicic acid and the fermented fruit extracts, it was found that the extracts of pomegranate caused a reduction in the number of lesions obtained and supports the activity of pomegranate extracts in the treatment of breast cancer [23].

The in vitro studies conducted on cancer stem cells derived from MMTV-Wnt-1, pomegranate extract was found to inhibit the proliferation of cancer cell by arresting the cell cycle at an early phase and induced apoptosis of the cancer cells. Pomegranate extracts caused an elevation I the levels of the enzyme caspase 3 which aids in the apoptosis. Among the various extracts, ellagic acid and ursolic acid along with luteolin were found to cause the inhibition of cell proliferation. Also, pomegranate extracts showed promising results in the molecular studies conducted on the MCF-7 cells of breast cancer through the inhibition of proliferation of the cancer cells. In the MCF-7 cells, the anti-cancer activity was found to be due to the cell cycle arrest, down regulation of genes which proliferate the cancer cells and also through the upregulation of the genes which aids in the regulation of proliferation and apoptosis. Hence, pomegranate extracts are relevant in the treatment of breast cancer therapy in the cases which are relatively resistant to the existing agents of treatment [24, 25].

#### **4. Pomegranate and colon cancer**

Colorectal cancer is currently one of the most common diagnosed cancer in men and women and it manifests with the uncontrolled proliferating of the epithelial cells and the suppression of their apoptosis [26]. One of the major constituents of pomegranate, the ellagitannin urolithin A plays a key role in the inhibition of proliferation of colon cancer cells through cell cycle arrest and the inhibition of mitogen activated protein kinase signaling (MAPK) [27].

The action of ellagitannins and urolithin on the CYP1 enzymes is important as these enzymes lead to the activation of inactive carcinogens into active carcinogenic chemicals in colon cancer. In the cell line study using HT-29 colon cancer cells, the evaluation of activity of CYP1 enzyme by employing EROD assay (ethoxy resorufin-*O*-deethylase assay) showed a reduction of CYP1 enzymes which were induced in the cell line. The extracts were found to show selective inhibition of proliferation of the cells omitting the non-cancer cells in a dose dependent fashion. Further, ellagitannin and urolithin was found to cause an increase in the apoptosis of the cell lines resulting in a reduction of the cell colony [28].

From the animal studies conducted on rats which were induced with colon cancer using N-methylnitrosourea which caused an increase in antigens which were specific to colon cancer along with and increase in plasma levels of Bcl2 and TGF-β, it was found that pomegranate peel extracts caused a fall in the cancer specific parameters which were induced in the mice. The in vivo study further suggests the efficacy of pomegranate in the treatment of colon cancer through the inhibition of proliferation and increased apoptosis which was evident from the fall in CEA and CCSA-4 prostate cancer cell markers along with the down regulation of β-catenin genes which has a pivotal role in the advancement of colon cancer. The down regulation of the specific gene disrupts the signaling pathway involving Wnt/β-catenin [29, 30].

From the cell line studies using HCT116 and HT-29 colon cancer cell lines, pomegranate extracts comprising of punicalagin, ellagic acid and tannins showed a drastic antiproliferative activity which led to complete inhibition of proliferation depending on the dose. The extract was found to cause apoptosis in the selected cell lines. Further, the extracts were found to have effect on the colon cancer cells which were not metastatic. The cell line studies further cement the role of punicalagin, ellagic acid and pomegranate tannins in the cancer protective activity in colon cancer [31].

#### **5. Pomegranate and head and neck cancer**

Head and neck cancers are one of the prevalent type of cancer which usually includes squamous cell carcinomas found in the epithelial cells of the pharynx,

#### *Role of Pomegranate in the Management of Cancer DOI: http://dx.doi.org/10.5772/intechopen.97188*

larynx and the oral cavity [32]. Due to the underdeveloped methods of screening of the disease, the chances of predicting the cancer at an early stage is less. This condition further leads to the increase in number of people who are diagnosed at a late stage of disease progression. In the current scenario, the treatment strategy of the disease mainly involves chemoradiation and surgery. The therapeutic approach to head and neck cancer comes with the common side effects of mucositis and dermatitis. Pomegranate extracts were studied for its protective effect in ameliorating the side effects of the treatment. In the clinical setup of a cohort containing patients with head and neck cancer, it was found that the extracts reduced the extend of damage caused by radiation induced dermatitis as well as mucositis [33].

Radiation therapy is applied in the cancer therapy for a long time because of its ability to kill the tumor cells but this will also lead to the production of reactive oxygen species that will damage the normal adjacent cells. Pomegranate extract has been studied in the amelioration of cellular damage induced by these reactive oxidants. From a study conducted using the extracts from pomegranate fruit and seeds, it was found that the treatment with the extracts increased the levels of antioxidant and the enzymes which has antioxidant property. Further the extracts were found to cause a decline in the lipid peroxidation levels suggesting the protective effect of pomegranate extracts in the cancer treatment as an adjuvant to reduce the unwanted side effects [34].

Other than in chemoradiation, pomegranate fruit extracts rich in punicalagin has been found useful in acting as a protective agent for the skin fibroblast cells namely the SKU-1064 from possible apoptosis due to UV-A and UV-B exposure. The extracts were found to suppress the NF- κB activation and through the downregulation of caspase-3 which is proapoptotic. Further studies found an increase in DNA repair through the increase in G0/G1 phase [35]. All these findings further supports the fact that pomegranate extracts can be applied in the treatment of cancer as an adjuvant also as a protective for radiation induced cellular damage.

#### **6. Pomegranate and lung cancer**

Lung cancer is one of the leading causes of death related to cancer worldwide in both men and women. Cigarette smoking is attributed to e the major cause of the condition. Along with lung cancer, cigarette smoke causes an increase in oxidative stress and DNA damage. From the animal studies conducted on the formation of lung nodules associated with lung cancer and other cancer related factors like the attenuation of mitosis and the levels of hypoxia inducible factor-1α or HIF1 α because of cigarette smoke, it was found that pomegranate juice supplements wee able to reduce the formation of lung nodules which is a common observation in the case of cigarette smoke exposure along with the reduction of mitosis and HIF-1 α [36].

Further, pomegranate fruit extract treatment in the cell line study using human carcinoma cell associated with lung cancer namely the A549 cells showed an inhibition of the markers of cell proliferation and angiogenesis such as MAPK, NIF-kappa B and PI3K/Akt. The treatment with the extracts further arrested the growth of tumor cells. Thus, pomegranate may be useful as a chemo preventive or as a chemotherapeutic agent against cancer affecting lungs [37]. Methotrexate is a widely used chemotherapeutic agent but it causes injuries in the lung cells due to oxidative stress. In the animal studies employed to study the effect of pomegranate extracts on the protective action against the lung injury caused

due to methotrexate, it was found that the use of pomegranate extracts as a prophylactic significantly reduced the total oxidant status and the oxidative stress index along with elevating the total antioxidant capacity. This in turn shows the application of pomegranate extracts as an adjuvant as well in the therapy of lung cancer [38, 39].

#### **7. Pomegranate and skin cancer**

Pomegranate extracts from seed, peel and the whole fruit have been proven to be beneficial in the treatment of many cancer treatments. Skin cancer is the most common cancer among the Caucasian population and it varies depending on the type of cells affected. UV radiation is the major cause of skin cancer since it initiates and promotes tumor [40]. The in vivo and in vitro studies has shown the efficacy of pomegranate as a protectant in the UVB radiation induced skin damage. The oral treatment of pomegranate juice and extract in the Fitzpatrick II-IV skin type showed the possibility of enhancement in the protective from UV damage since it is able to increase the threshold of the UV dose required to cause erythema of skin [41].

The oil extracted from seed of pomegranate fruit was studied on animals as a topical prophylactic in the mice which was induced with skin cancer using 12-*O*-tetradecanoylphorbol 13-acetate and 7,12-dimethylbenzanthracene and it was found that the pre-treated animals had a significant less incidence of skin cancer which tells the protective effect of pomegranate in skin cancer [42]. From the studies conducted on the human skin fibroblasts SKU-1064 which were irradiated with UV, it was found that the pomegranate extracts rich in punicalagin was able to prevent the skin cell death showing the effect of pomegranate as a topical protective agent against skin cancer [35].

#### **8. Conclusion**

Pomegranate and the products derived from it has been proven to show various medicinal properties. Even though it has been in use in various traditional medical folklore since ages, the medicinal property of pomegranate is not explored much to be of use in the current medical scenario. Pomegranate is still being used as just a fruit and from the studies which are conducted so far on the fruit, it is to be noted that the extracts of the fruit rather than the whole fruit as such possesses many medicinal properties.

The role of pomegranate in the therapy of cancer as such and as an adjuvant in therapy is explored very less as there are very few studies has been conducted on humans, even though there are a handful of studies which are conducted on animal models or cell line studies which deems the fruit and its extracts effective in the therapy of cancer. The studies conducted so far shows the potency of pomegranate and its components in the treatment of cancer relating to prostate, breast, head and neck, colon, lungs and skin or as an adjuvant in the treatment to minimize the unwanted side effects. The various components of pomegranates because of its antioxidant and anti-inflammatory property can be applied to various treatment strategies in numerous types of cancer in one way or the other.

Hence it can be concluded that pomegranate extracts can be made to much use for humans in improving the treatment strategies in turn improving the quality of life, for which there has to be more human, animal and cell line studies so that the complete potency of pomegranate can be uncovered.

*Role of Pomegranate in the Management of Cancer DOI: http://dx.doi.org/10.5772/intechopen.97188*

#### **Author details**

Amulya Thotambailu1 \*, Deepu Cheriamane2 , Manjula Santhepete3 , Satheesh Kumar Bhandary4 , Jiju Avanippully5 and Prakash Bhadravathi6

1 Department of ENT, JSS Medical College and Hospital, JSS Academy of Higher Education and Research, India

2 Department of Pulmonary Medicine, Sridevi Institute of Medical Sciences, Tumkur, India

3 Department of Pharmacology, JSS College of Pharmacy, Mysuru, JSS Academy of Higher Education and Research, Mysuru, India

4 ENT, NITTE (Deemed to be University), India

5 JSS College of Pharmacy, Mysuru, India

6 JSS Academy of Higher Education and Research, India

\*Address all correspondence to: amulyathotambailu@gmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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*Role of Pomegranate in the Management of Cancer DOI: http://dx.doi.org/10.5772/intechopen.97188*

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#### **Chapter 4**

## Vasculoprotective and Neuroprotective Effects of Various Parts of Pomegranate: In Vitro, In Vivo, and Preclinical Studies

*Maria Trapali and Vasiliki Lagouri*

### **Abstract**

Pomegranate (*Punica granatum* L.) is one of the oldest edible fruits in the Mediterranean area and has been used extensively in the folk medicine. Popularity of pomegranate has increased especially in the last decade because of the health effects of the fruit. Polyphenols, represent the predominant class of phytochemicals of pomegranate, mainly consisting of hydrolysable tannins and ellagic acid. Pomegranate is a rich source of the ellagitannin punicalagin, which has aroused considerable interest in pomegranate fruit as a new therapeutic agent in recent years. Most studies on the effects of pomegranate juice have focused on its ability to cure diabetes and atherosclerosis. The present review summarizes some recent studies on the vasculoprotective and neuroprotective effect of various parts of pomegranate and its main compounds especially hydrolysable tannins ellagitannins, ellagic acid and their metabolites. The in vitro and in vivo studies, showed that the whole parts of pomegranate as well as its main components had a positive influence on blood glucose, lipid levels, oxidation stress and neuro/inflammatory biomarkers. They could be used as a future therapeutic agent towards several vascular and neurodegenerative disorders such as hypertension, coronary heart disease and Alzheimer.

**Keywords:** pomegranate, ellagic acid, punicalagin, urolithins, cardiovascular disease, CNS, in vitro, in vivo, pre-clinical trials

### **1. Introduction**

Free radical reactions occur naturally in the human body. An over-production of these reactive species due to oxidative stress can cause oxidative damage to biomolecules and the development of chronic diseases such as aging, coronary heart disease and cancer [1]. The harmful action of free radicals can be inhibited by antioxidant substances which scavenge them and detoxify the organism. Current research has confirmed that dietary antioxidants play an important role in the prevention of cardiovascular diseases and cancers, neurodegenerative diseases and inflammation [2]. Pomegranate (*Punica granatum* L.) is one of the oldest edible fruits in the Mediterranean area and has been used extensively in the folk medicine. Popularity of pomegranate has increased in the last years because of anti-microbial, anti-viral, anti-cancer, anti-oxidant and anti-mutagenic effects of the fruit [3–5]. Polyphenols, are the main phytochemicals of pomegranate fruits, mainly consisting of hydrolysable tannins, gallotannins, ellagitannins and ellagic acid (EA). It has been found to exhibit antimutagenic, antiviral, whitening of the skin and antioxidative properties [6, 7]. Pomegranate fruit is composed of three different parts: the seeds, the arils and the peels. The therapeutic properties have been reported mostly for pomegranate juice [8–11] however, increasing literature was found lately reporting the inhibition of lipid peroxidation of pomegranate peels and seeds [4, 12, 13].

Even a small number of clinical trials in humans have been reported until now, the results showed positive effects of pomegranate extracts on various vascular diseases.

#### **2. Phytochemical components related to activity**

Ellagitannins (ETs) are esters of hexahydroxydiphenic acid (HHDP) and a polyol, usually glucose or quinic acid that when they are hydrolyzed transform through lactonization to the component ellagic acid [14] (**Figure 1**).

The variability in the chemical structures among ETs is associated with different physico-chemical properties, hydrolytic reactions, and biological activity in vivo [15]. The important structural diversity of ET structure is due to the different possible extent of galloylation and formation of aromatic C-glycosides, the number of intramolecular C-C coupling of galloyl groups and hydrolytic cleavage of galloylderived aromatic rings, the level of dehydrogenation, and oligomerization [16].

Ellagitannins and ellagic acid with anti-inflammatory and vasculoprotective effects are transformed by the gut microbiota to produce urolithins, bioavailable metabolites [17, 18] (**Figure 2**). There is, however, a large variability in health effects and can be associated with the different polyphenol glucuronide metabolic profiles. Differences in urolithin production, both quantity and chemical type, could explain, at least partly, the large variability in the health effects observed in vivo.

The effects of components of the pomegranate e.g. ellagic acid (EA) are also focusing on its potential protective action towards several neurodegenerative disorders. EA has been investigated as multi-target pharmacological drug on CNS in a review analysis [19]. Pomegranate metabolites such as urolithins prevented β-amyloid fibrillation in vitro and especially methyl-urolithin B (3-methoxy-6H-dibenzo [b, d] pyran-6-one), had a protective effect in *Caenorhabditis elegans* post induction of amyloid β(1–42) induced neurotoxicity and paralysis [20].

Urolithin A (UA) allayed hypoxia/reoxygenation abuse in myocardial cells, decreased myocardial cell death in mice after ischemia/reperfusion. UA enhanced antioxidant quantity in cardiomyocytes following hypoxia/reoxygenation reducing

#### **Figure 1.**

*Basic structures of ellagitannins: (A) HHDP acid (R radical); (B) galloyl unit (G radical); (C) ellagic acid.*

*Vasculoprotective and Neuroprotective Effects of Various Parts of Pomegranate: In Vitro, In Vivo… DOI: http://dx.doi.org/10.5772/intechopen.96680*

**Figure 2.**

*Gut microbiota metabolism of ellagitannins and ellagic acid.*

myocardial apoptosis [21]. The flavonoids naringin and narirutin have a significant beneficial effect in reducing diastolic blood pressure, in patients with hypertension [22]. Human umbilical vein endothelial cells (HUVECs) were pretreated with ellagic acid and then incubated with oxidized low-density lipoprotein (oxLDL). The results indicated inhibition of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, enhancing cellular antioxidant defenses, and attenuating oxLDL-induced Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) up-regulation and endothelial nitric oxide synthase (eNOS) downregulation. Lectin-like oxidized LDL (oxLDL) receptor-1 (LOX-1, also known as OLR-1, is a class E scavenger receptor that mediates the uptake of oxLDL by vascular cells. LOX-1 seems to represent an attractive therapeutic target for the treatment of human atherosclerotic diseases [23]. Adipocyte cells were pretreated with punicalagin and ellagic acid and that caused inhibition of lipolysis reducing MAO activity [24].

Urolithin C, a combination of urolithins A and B metabolites of pomegranate and ellagic acid also reduced cholesterol accumulation in the human monocytic cell line THF-1-derived macrophages, but were unable to promote cholesterol outflow. Atherosclerotic processes can be attenuated by urolithins, but future human

intervention tests are needed to see if it translates in vivo [23]. The ability of punicic acid (PUA) to modulate peroxisome proliferator-activated receptor PPAR activity was determined in 3 T3-L1 pre-adipocytes. PUA activates PPAR, increases PPAR responsive gene expression and ameliorates diabetes and inflammation [25].

#### **2.1 In vitro studies**

PJ concentrate reduced the activation of redox-sensitive genes (ELK-1 and p-JUN) and increased eNOS expression in cultured human coronary artery endothelial cells (EC) exposed to high shear stress in vitro [26]. In vitro study showed that pomegranate leaf, seed and juice repressed cholinesterase activity in a dose dependent manner. Pomegranate juice had also protective effects against hydrogen peroxide induced toxicity in the *Artemia salina* (a species of brine shrimp) and HepG2 models (in vitro model system for the study of polarized human hepatocytes), antiproliferative activities in HeLa and PC-3 cancer cells inhibiting COX-2 and MAO enzymes [27].

Microglial cells are the resident macrophages of the CNS. The immortalized murine microglial cell line BV-2 has been used frequently as a substitute for primary microglia. Urolithin B inhibited the production of NO and pro-inflammatory cytokines, inhibited NF-κB activity by reducing the phosphorylation and degradation of a nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, IκBα. In addition, urolithin B suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERK), and Protein kinase B Akt, and enhanced the phosphorylation of AMPK, which is associated with antiinflammatory and antioxidant processes [28, 29]. In another study, lipopolysaccharide LPS-treated cultured astrocytes and microglial BV-2 cells were investigated for anti-neuroinflammatory effects of punicalagin (PUN). It was found that PUN inhibits LPS-induced memory impairment via anti-inflammatory and anti-amylogenic mechanisms through inhibition of nuclear factor kappa-light-chain-enhancer of activated B cells NF-κB activation [30]. The above results may be a solution to Alzheimer Disease [31].

#### **2.2 In vivo studies**

Clinical studies in hypertensive and/or obese patients receiving pomegranate juice have shown a reduction in systolic and diastolic blood pressure [32–36] and a concomitant increase in high density lipoprotein (HDL) cholesterol. Juice intake also led to a significant reduction in the by-products of fat peroxidation and protein and inflammatory biomarkers. Patients taking pomegranate-containing nutrient supplements had lowered systolic and diastolic blood pressure levels but the cardiovascular risk did not recover [37].

A number of clinical trials in humans proved the positive effects of pomegranate juice in the protection of central nervous system (CNS). Maternal pomegranate juice absorption in pregnancies with intrauterine growth restriction (IUGR) showed differences in the infant brain and structure [38].

#### **2.3 Preclinical studies**

When PJ was given in diabetic rats it was observed decreased blood glucose, lipid levels, and inflammatory biomarkers [39]. In another study using obese Zucker rats, intake of pomegranate juice (PJ) or fruit extract PFE caused a decrease of inflammation factors and increase of plasma nitrate and nitrite (NOx) [40]. In a study involving diabetic rats, they were given pomegranate seed powder (PS).


*Vasculoprotective and Neuroprotective Effects of Various Parts of Pomegranate: In Vitro, In Vivo… DOI: http://dx.doi.org/10.5772/intechopen.96680*

#### **Table 1.**

*Vasculoprotective and neuroprotective effects of pomegranate and their substances/metabolites in in vitro and in vivo pre-clinical studies.*

Increased blood cholesterol, LDL and HDL lipoprotein were found [39, 41] while systolic blood pressure, angiotensin-converting enzyme coronary activity decreased [42]. Pomegranate peel (PPE), flower (PFE) and seed (PSO) given in obese mice decreased fasting blood glucose, improved insulin sensitivity, increased levels of the anti-inflammatory cytokine interleukin-10 [43] and activated peroxisome proliferator-activated receptor gamma (PPARγ) [25]. PPARγ, a ligand-activated transcription factor, has a role in various cellular functions as well as glucose homeostasis, lipid metabolism, and avoidance of oxidative stress. Pigs with hypercholesterolemia were given a pomegranate extract which caused reduction of systemic oxidative stress [33]. Pomegranate supplementation also exhibits cardiovascular protection improving cardiac hypertrophy in cigarette smoke in sight animals [11].

Preclinical trials in animal models added research results to the positive effects of pomegranate in CNS. In a rat model of Parkinsonism induced by rotenone, pomegranate juice treatment resulted in protection against oxidative destruction and improvement of neuronal durability [44]. Besides, in a rat model of maternal inflammation, pomegranate juice caused inhibition of fetal brain apoptosis, neuronal nitric oxide synthase, and nuclear factor-κB activation [45] (**Table 1**).

Methods used are extensively described in literature (e.g. [21, 37, 46–50]).

#### **3. Possible therapeutic applications**

The in vitro and in vivo studies showed that the whole parts of pomegranate as well as its main components such as hydrolysable tannins, ellagic acid and urolithins had a positive influence on blood glucose, lipid levels, oxidation stress and neuro/ inflammatory biomarkers.

#### **4. Future perspective and recommendations**

The reviewed studies emphasize the potential benefits and suggest of a wider use of pomegranate and its components as dietary supplements or as adjuncts in the treatment of vascular and neurodegenerative diseases such as hypertension, coronary heart disease, peripheral artery disease and Alzheimer disease.

### **Conflict of interest**

The authors declare that there are no conflicts of interest regarding the publication of this chapter.

*Vasculoprotective and Neuroprotective Effects of Various Parts of Pomegranate: In Vitro, In Vivo… DOI: http://dx.doi.org/10.5772/intechopen.96680*

### **Author details**

Maria Trapali1 \* and Vasiliki Lagouri<sup>2</sup>

1 Laboratory of Chemistry, Biochemistry, Cosmetic Science, University of West Attica, Aigaleo, Greece

2 Institute of Chemical Biology, National Hellenic Research Foundation, Athens, Greece

\*Address all correspondence to: mariatrapali66@yahoo.gr

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 5**

## Could Pomegranate Fight against SARS-CoV-2?

*Sally Elnawasany*

#### **Abstract**

Pomegranate, *Punica granatum* L., is an authentic, generous fruit which is cultivated in many parts of the world for thousand years. The divine fruit was born from nature to provide humanity with its effluent benefits for life and health. Through the ages, Pomegranate occupied an eminent place in ayurvedic medicine. It was prescribed for treatment of parasitic infection, diarrhea, and ulcers. Pomegranate wealth of prolific pharmacological activities makes it a rich culture for multiple studies in recent years. It will not be surprising if Pomegranate provides humans with a possible help in SARS-CoV-2 pandemic. The enemy that has raided the world since the end of 2019.

**Keywords:** ayurvedic medicine, phytochemicals, pomegranate, SARS-CoV-2

#### **1. Introduction**

Pomegranate (*Punica granatum* L.) is a common authentic fruit that is consumed for its health benefits in the globe. It contains many phytochemical constituents mainly Phenolic compounds which are responsible for most of its pharmacological properties [1, 2]. Several studies roamed in the Pomegranate field for its therapeutic benefits; anti- inflammatory, anti -oxidant, anti-cancer, anti- viral and immune modulation activities [3]. The fact that put pomegranate on the top of phytochemical agents with possible anti SARS-CoV-2 potential. Which has been attacking the earth for over a year. It is one of Coronaviruses, member of the subfamily Coronavirinae in the family Coronaviridae and the order Nidovirales [4].

#### **2. Severe acute respiratory syndrome coronavirus-2, SARS-CoV-2**

Corona viruses are wide group of viruses of humans as well as some animals. The clinical impact is ranged from mild to severe respiratory disease. In the last two decades, the world faced two aggressive coronaviruses: severe acute respiratory syndrome coronavirus (SARS-COV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-COV) in 2012 [4]. At the end of 2019, SARS-CoV-2 was reported in China, as an abnormal highly contagious viral pneumonia. Then shortly, the virus invaded the whole world [5, 6]. SARS-CoV-2 is an enveloped positive-sense single stranded RNA virus. It consists of four subunits, spike (S) glycoprotein, small envelope (E) glycoprotein, membrane (M) glycoprotein and nucleocapsid (N) protein [7]. Spike S protein with its two subunits, S1 and S2 is responsible for epithelial cell entry after its attachment to Angiotensin Converting Enzyme 2, ACE2 receptors which is widely present in the respiratory tract and other parts of the body [8, 9]. While surface S1 subunit (specifically at receptor-binding domain, RDB region) attach to ACE2 receptor, transmembrane subunit (S2) starts membrane fusion between the virus and epithelial cell and begins endocytosis. This process is enabled by the two host cell enzymes; furin and transmembrane serine protease 2 (TMPRSS2) that cleaves S glycoprotein at S1/S2 [10, 11]. Then SARS-CoV-2 replicates and spreads down to the airways and occupies alveolar epithelial cells. Viral replication induces Intense immune response (Cytokine storm syndrome) with subsequent acute respiratory distress syndrome and respiratory failure, the main cause of death [12]. Treating SARS-CoV-2 infection is not easy, as we have not only to fight the virus and manage its respiratory sequalae, but we need to downregulate the hyper stimulated immune response as well. For this war, many agents have been recruited in different ways. Starting from Inhibition of virus entry as Umifenovir (Arbidol) that interferes with interaction between the viral S protein and ACE2 and block membrane fusion [13, 14]. Chloroquine and hydroxychloroquine (two drugs of plant origin) are also thought to inhibit viral entry but with controversial results [15, 16]. Using soluble recombinant hACE2, specific monoclonal antibodies to occupy ACE2 receptors is another method to counter act viral entry [17, 18]. Inhibition of virus replication is another modality for treatment. There are numerous trials on remedesivir [15], favipiravir [19], ribavirin, lopinavir and ritonavir to inhibit viral replication [20]. Since SARS-CoV-2 over stimulates the immune response causing what is called, cytokine storm syndrome [21]. Immune modulation is a promising target for treatment. Dexamethasone decreased mortality in mechanically ventilated and oxygen receiving patients [22]. Plasma from recovered patients, convalescent plasma-derived hyperimmune globulin and monoclonal antibodies targeting SARS-CoV-2 were also tried in many trials [23–26]. Interleukin-6 (IL-6) has an important role in the inflammatory response. Tocilizumab, interleukin-6 (IL-6) receptor-specific antibody downregulated the immune response in small trials [27, 28]. Moreover, inhibition of pro inflammatory Complement 5 by Eculizumab, a specific monoclonal antibody, helped to decrease pulmonary oedema in severe COVID-19 patients [29]. Interferon plays a role in reducing of viral replication, type I interferons provide a treatment options in COVID-19 infection [30, 31]. Protein kinases inhibitors as Baricitinib, a reversible Janus-associated kinase (JAK)-inhibitor can help in SARS-CoV-2 treatment through its anti-inflammatory, anti-viral and antifibrotic properties [32]. Baricitinib attenuated cytokine signaling in COVID-19 immune response. It also interfered with viral cell entry [33]. In another study, it improved with corticosteroids the respiration in SARS-CoV-2 pneumonia [34]. In addition, the Abl tyrosine kinase inhibitor (ATKI), imatinib was found to block viral fusion through attachment to receptorbinding domain (RBD) of SARS-CoV-2 spike protein [35]. In spite of all the previous treatment modalities, there is no proven curative agent for SARS-CoV-2 infection [36]. Which necessitates a continuous and hard search for new therapeutic agents including natural agents.

#### **3. Could pomegranate fight against SARS-CoV-2?**

#### **3.1 Anti-viral action of pomegranate**

Pomegranate attenuates many viruses [37]. Polyphenols and ellagic acid were proved to neutralized envelope virus via binding to the envelope lipid or sugar moieties [38]. Pomegranate juice succeeded to prevent Human immune deficiency virus-1 (HIV-1) cell entry by blocking CD4 and coreceptors CXCR4/CCR5

*Could Pomegranate Fight against SARS-CoV-2? DOI: http://dx.doi.org/10.5772/intechopen.96423*

binding [39]. Ellagitannins of Pomegranate extract; punicalagin, punicalin and ellagic acid blocked the HCV NS3/4A protease activity in an in vitro study [40]. Furthermore, the activity of adenovirus was suppressed by Pomegranate peel ethanol extract on HeLa cell line. The 50% inhibitory concentration (IC50) and 50% Cytotoxicity Concentration (CC50) were 165 ± 10.1 and 18.6 ± 6.7 μg/ml, respectively [41]. In addition, Pomegranate juice and pomegranate polyphenol extract reduced viral titer of noroviruses with other foodborne viral surrogates [42]. A viricidal effect of Pomegranate powder extract with 800 μg/ml polyphenols was clarified. When the titer of influenza virus (PR8 (H1N1), X31 (H3N2), and a reassortant H5N1 virus of human isolate lowered by 3log in 5 min treatment at room temperature. This effect was explored by electron microscopy when disruption of viral structure appeared [43]. Moreover, the replication and agglutination of chicken RBC's by influenza virus was inhibited by Punicalagin, a phenol in pomegranate extract. Synergistic effect was noticed in oseltamivir combination [44]. The anti Influenza mechanism was emphasized in another study where Pomegranate peel ethyl alcohol extract (PPE) inhibited the influenza virus adsorption and replication though attenuation of viral polymerase activity and protein expression [45].

#### **3.2 Immune modulatory action of pomegranate**

Mast cells and basophils have a crucial role in inflammatory and immune response [46]. These cells release pro-inflammatory cytokines TNF-α, IL-6, IL-8, histamine which initiate acute- and late-phase inflammatory response [47]. Cytokine expression is induced by many pathways such as extra-cellular signalregulated kinase (ERK), and c-Jun N-terminal kinase (JNK) and Nuclear factor (NF)-κB [48–50]. Immune modulation action of pomegranate was confirmed in many studies. Pomegranate fruit extract strongly attenuated phorbol-12-myristate 13-acetate plus calcium inophore A23187 (PMACI) induced inflammatory gene expression and reduced the release of interleukin (IL)-6 and IL-8 in the myeloid pre-cursor cell line KU812 cells. Through its action on c-jun N-terminal kinase (JNK), extracellular-regulated kinase (ERK) and Neucular factor Kappa β (NF-κB) dependent pathways [51]. NF-κB signaling stimulation is mediated by IL-1β binding to its specific cell surface receptor that activates IKKs with subsequent phosphorylation and degradation of IκB. This cascade was suppressed in human chondrocyte by pomegranate extract. Which interfered with the mRNA and protein expression of IL-6 and downregulated the activation of NF-κB/p65. Through inhibition of the IL-1β-mediated phosphorylation of IKKβ, expression of IKKβ mRNA and degradation of IκBα [52]. Moreover, in another in vitro study, Pomegranate flower (PFE) ethanol extract reduced IL-6, IL-1β and TNF-α production with IC50 valueof 48.7, 71.3 and 62.5μg/mL respectively, in lipo-poly saccharides (LPS) -induced RAW264.7 cell macrophage. This effect was attributed to inhibition of phosphorylation of mitogen-activated protein kinase (MAPK) subgroups, extracellular signalregulated kinase (ERK), c-Jun N-terminal kinase (JNK) and P38 and translocation of the NF-B p65 subunit [53]. Pomegranate peel extract decreased the secretion of CXCL8 in both Caco-2 cells and colonic explants. Furthermore, it attenuated the expression of IL 1A, IL 6 and CXCL8 in lipopoly saccharide, LPS stimulated colonic tissues at a concentration of 5 g/ml [54].

#### **3.3 Anti-tyrosine kinase action of pomegranate**

Janus kinase (JAK) is a member of the non-receptor tyrosine kinase family. It triggers many inflammatory signaling pathways like signal transducer and activation of transcription (STAT) that induce chemotaxis of inflammatory cells such

as mast cells, T cell, B cells, macrophages [55]. Baricitinib is a Janus kinase (JAK) inhibitor and is a numb-associated kinase NAK inhibitor which attenuates AP2 associated protein kinase-1 (AAK1), the protein that promotes viral endocytosis [56, 57]. Fortunately, Pomegranate shares Baricitinib its janus kinase inhibitory action. The fact that introduces Pomegranate as a possible treating agent of SARS-CoV-2. This action was highlighted in a study where Pomegranate leaf extract antagonized Janus Kinase1 (JAK1) enzyme activity in macrophage raw cells [58]. In another study, among ellagitannins containing fruits, pomegranate was the superior in JAK2 inhibition [59].

#### **3.4 Anti-converting enzyme (ACE) action of Pomegranate**

The renin-angiotensin-aldosterone system, RAAS organizes blood pressure, fluid balance and controls the vascular response to inflammation [60]. Imbalance in that system induces hypertension, fluid retention, and inflammatory and thrombotic complications [61]. Juxtaglomerular apparatus of the kidney secretes renin which acts on angiotensinogen to form Angiotensin I (A1). Angiotensin-converting enzyme (ACE) breaks AI to AII. Angiotensin II is the main controlling agent of RAAS through stimulation of type 1 receptor (AT1 receptor) with subsequent vasoconstriction, water retention and inflammation. While The type 2 receptor, ATR2 counteract these effects [62]. ACE2 counterbalance ACE actions. It breaks down AI into angiotensin 1-9(A1-9), and AII into angiotensin 1-7(A1-7) which has vasodilator and anti-proliferative action [63]. The renin-angiotensin system is claimed to induce severe acute lung injury in SARS-CoV-2 infection and ACE2 protects against acute lung failure and its deficiency is associated with lung damage [64]. Binding of SARS-Cov-2 to ACE2 receptor attenuates ACE2 action with subsequent lung damage [65]. On that basis, soluble ACE2 was supposed to be a possible approach for coronavirus infection [66]. It was speculated that, The use of ACE Inhibitors is associated with increased concentration of angiotensin I which upregulates ACE2 [67]. It is ambiguous, whether this postulate increases the probability of SARS-CoV-2 infection. Or ACE2 upregulation will be beneficial for counterbalance the ACE2 virus-induced downregulation with improvement of lung defense [65]. Although the role of Angiotensin converting enzyme, ACE inhibitors in SARS-CoV-2 infection is controversial, Pomegranate has the potential of ACE inhibition and may help in this battle. *Punica granatum* juice extract lowered ACE level and mean arterial blood pressure. When it was given in a dose of (PJ- 100 mg/kg and 300 mg/kg: p.o.) in angiotensin-II treated rats for 4 weeks [68]. Similar effect was emphasized in a parallel study. When Pomegranate peel extract was administered to female rats for 30 days. It inhibited coronary angiotensin-converting enzyme (ACE) activity and oxidative stress [69]. In a clinical trial, Pomegranate juice reduced serum ACE activity and systolic blood pressure in hypertensive patients when it was consumed for 2 weeks at a dose of (50 ml, 1.5 mmol of total polyphenols per day) [70].

#### **3.5 Anti-SARS-CoV-2 action of pomegranate**

Pomegranate potentials against SARS-CoV-2 infection have been investigated in many studies.

#### *3.5.1 Anti-SARS-CoV-2 action of pomegranate*

In a Computational study, Pomegranate peel extracts components; ellagic acid, gallic acid and specially punicalagin, punicalin showed promising anti SARS-CoV-2 activity through interaction with SARS-CoV-2 spike glycoprotein, angiotensin

*Could Pomegranate Fight against SARS-CoV-2? DOI: http://dx.doi.org/10.5772/intechopen.96423*

converting enzyme 2, furin and transmembrane serine protease2. They formed more stable complexes with amino acid residues at the active sites of the selected protein targets in comparison to positive controls (umifenovir, lopinavir, camostat) with more significant binding affinity. Punicalin showed the most potent interaction with the S glycoprotein with free binding energy of −7.406 kcal/mol. All Pomegranate components ligands exerted a significant binding affinity at the ACE2 predicted active site. Furthermore, they formed the most stable complexes with furin. Amazingly, Punicalagin and punicalin strongly interacted with TMPRSS2 amino acid residues at the predicted active site by binding energy values of −7.358 and − 8.168 kcal/mol, respectively with higher affinity for the target protein than camostat (−7.069 kcal/mol) [71]. In an in vitro study, Pomegranate juice reduced the infectivity of SARS-Cov-2 and influenza virus in VeroE6 cells [72]. In another study, Pomegranate peel extract showed an ability to block the binding between SARS-CoV-2 Spike glycoprotein and the human Angiotensin-Converting Enzyme 2 (ACE2) receptor, furthermore, it downregulated the activity of the virus bind 3-chymotrypsin-like cysteine protease (3CLPro) (an enzyme which is important for viral replication) [73].

#### *3.5.2 Anti-SARS-CoV-2 action of natural compounds that are found in pomegranate*

In a virtual study, pedunculagin, tercatain, and castalin (hydrolysable tannins) showed an ability to bind (3CLPro) catalytic site that is involved in SARS-CoV-2 replication. Which sheds the light on tannins as possible anti SARS-CoV-2 agents [74]. Other virtual study investigated the action of natural compounds on SARS-CoV-2 Spike protein, viral Protease and RNA-dependent RNA polymerase and host cell protease TMPRSS2. Triterpenoids was found to be the superior in blocking the Spike protein binding site of SARS-CoV-2 [75].

### **4. Conclusion**

Pomegranate is still surprising the world by its great therapeutic benefits. This chapter highlights the anti-SARS-CoV-2 potentials of Pomegranate. Where Antiviral, immune modulation, tyrosine kinase and ACE inhibition actions, all enable Pomegranate to fight in this war. Further future studies are needed to confirm the utility of Pomegranate in treating SARS-CoV-2 infection.

#### **Conflict of interest**

I confirm that there are no conflicts of interest.

*Pomegranate*

#### **Author details**

Sally Elnawasany Tropical Medicine, Tanta University, Egypt

\*Address all correspondence to: elnawasany\_s@hotmail.com

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Could Pomegranate Fight against SARS-CoV-2? DOI: http://dx.doi.org/10.5772/intechopen.96423*

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### Section 3
