Therapeutic Potential of Punica granatum and Isolated Compounds: Evidence-Based Advances to Treat Bacterial Infections

Punica granatum Linn has been known for its nutritional and medicinal value since ancient times and is used in the treatment of various pathologies owing to its antibacterial properties. This review reports the results of the most recent studies on the antibacterial effects of P. granatum and its isolated compounds on bacteria of clinical interest. A search in the PubMed, Scopus, Science Direct, and Science Citation Index Expanded (Web of Science) databases was performed, which included articles that evaluated the antibacterial activity of P. granatum extracts and excluded articles that analyzed other microorganisms or nonpathogenic bacteria, as well as theses, dissertations, duplicate articles, and those not fully available. The literature suggests that P. granatum extracts can act on bacteria, such as methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA), Streptococcus mutans, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In addition, fruit peel was the most commonly used pharmacogen and methanol, ethanol, and water were the most common solvents for the extraction of bioactive compounds. The antibacterial potential of the methanolic extract of pomegranate peel could be attributed to the presence of active compounds, such as 5-hydroxymethylfurfural, punicic acid, gallic acid, and punicalagin. Thus, there is evidence that these plant extracts, having high polyphenol content, can disrupt the bacterial plasma membrane and inhibit the action of proteins related to antimicrobial resistance. P. granatum shows antibacterial activity against Gram-positive and Gram-negative bacteria, with great potential against multidrug-resistant strains. Further research is needed to clarify the mechanism of action related to this biological activity and investigate the isolated substances that may be responsible for the antibacterial effects.


Introduction
Te multidrug-resistant (MDR) bacteria are creating a serious challenge to treat diseases, and the essential oils from medicinal plants are efective natural products for resistant pathogen bacteria [1][2][3][4].Medicinal plants are widely used as herbal medicines, and the World Health Organization (WHO) recognizes that much of the population of developing countries depends on traditional medicine for their primary care; 80% of this population uses traditional practices in their basic health care and 85% uses plants or their preparations.Since the Declaration of Alma Ata in 1978, in which the use of medicinal plants and herbal medicines for prophylactic, curative, and palliative purposes was recognized, the WHO has encouraged the use of medicinal plants for healthcare [5].
Natural products have proven to be alternative and potential sources of synthetic drugs.Studies have shown that crude extracts or purifed chemical constituents of various medicinal plants are often more efective than synthetic antioxidants [6].P. granatum (pomegranate) has been widely used in the food and pharmaceutical industries for a variety of applications and as a source of antioxidants to combat autoxidation-induced pathologies [7,8].Punica granatum Linn, belonging to the family Punicaceae, is well known for its nutritional and medicinal value [9].Approximately, 50% of the total weight of fruits corresponds to the peel, which is an important source of bioactive compounds, such as phenolics, favonoids, ellagitannins, and minerals, which primarily include potassium, nitrogen, calcium, phosphorus, magnesium, sodium, and complex polysaccharides [10].
In addition to its nutritional benefts, the fruit is used empirically for the treatment of various diseases, such as acidosis, dysentery, microbial infections, parasitic infections, hemorrhage, and respiratory pathologies [9,11].Te pharmacological properties of pomegranate have a long history, and recent decades have seen a growing interest in its therapeutic efects [6].
Research has evidenced that pomegranate extracts can be used as natural alternatives for the treatment of various diseases, including bacterial and viral infections [12].Te antimicrobial potential of P. granatum extracts can be attributed to its high content of hydrolyzable polyphenols, consisting mainly of gallotannins and ellagitannins, such as punicalagin, punicalins, and ellagic acid [9].
As they provide low-cost therapeutic potential, there is considerable interest in the use of plant products as an alternative method to control pathogenic microorganisms and many natural product compounds have been observed to target pathogenic bacteria [7].
Tere is still much to explore regarding the antibacterial activity of this natural product, for instance, to improve the existing technologies or make them more accessible through the development of simpler, cheaper, and equally efcient technologies.Tese must utilize raw materials found in lessdeveloped regions, as some of the challenges in treating bacterial infections are the high cost of treatment, bacterial resistance, and inaccessibility of remote locations.
Terefore, this study aimed to conduct a literature review of the antibacterial efects of P. granatum and its isolated compounds on bacteria of clinical interest by synthesizing data on the production of extracts, frequently used evaluation methods, and the main results described in these studies.It also provides direction for future investigations to expand the chemical and biological knowledge of this species and highlights its most promising antibacterial compounds that are worthy of further exploration.

Materials and Methods
In this study, we performed an integrative review of the literature according to the following steps: elaboration of the guiding question, search for primary studies, evaluation of primary studies, data extraction, analysis, and synthesis of results and presentation.

Identifying the Research Question.
For the elaboration of the guiding question, the PICO strategy was used [18], in which "P" refers to the study population (bacteria of clinical interest); "I" is the intervention studied or the variable of interest (P.granatum used as the antibacterial agent); and "CO" is the context, which refers to the outcome of interest (antibacterial activity).Tus, the guiding question for this integrative review was "does the species P. granatum provide efective antibacterial action against bacteria of clinical interest?"2.2.Search Strategy.For the selection of articles in the literature, a search was performed using the following: PubMed, Science Direct, Scopus, and Science Citation Index Expanded (Web of Science); accessed: February 15 th -March 5 th , 2022, by the CAPES scientifc journals gateway.Te following controlled descriptors, keywords, and synonyms with Boolean operators were used for crossing the database: "Antibacterial Agents" OR "Agents, Antibacterial" OR "Antibacterial activity" AND "Pomegranate" OR "Pomegranates" OR "Punica granatum."Te data were collected between February and March 2022.

Inclusion and Exclusion Criteria.
Articles that evaluated the antibacterial activity of P. granatum extracts without language restrictions or time periods were included.Articles that analyzed other microorganisms or nonpathogenic bacteria and those that associated P. granatum with extracts of other natural products or antibiotics were excluded.In addition, theses, dissertations, duplicate articles, and articles that were not fully available were excluded.

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International Journal of Microbiology 2.4.Summarizing and Reporting the Results.Te selected articles were read in full for critical analysis and categorization.According to the adopted design, all were found to be in vitro preclinical trials [19].
Tese studies were also reviewed to extract data, such as the pharmacogen used, solvent extractor, bacterial strain, and antimicrobial susceptibility assays.Te results were analyzed to identify whether there was inhibition of bacterial growth expressed by inhibition zones (mm) and to determine the minimum inhibitory concentration (MIC) and/ or the minimum bactericidal concentration (MBC).Subsequently, the major compounds that may be responsible for the biological activity of the species were verifed.Subsequently, their molecular structures were illustrated with the help of ChemSketch software (ACD/Labs, version 2021.2.1).

Results and Discussion
Medicinal plants are widely recognized as safe treatment options, providing protective efects against a broad spectrum of toxic agents, whether natural or synthetic [20,21].Tese agents not only neutralize the negative efects of toxicants [22] but also play a crucial role in adjusting immunity-related genes, thereby boosting the body's defense against infections and infestations [23, 24] Among these, P. granatum has garnered extensive attention due to its constituents' involvement in various biological and pharmacological activities, contributing to overall health benefts [25].In this review article, we have focused on the direct action and immunomodulatory efects of pomegranate against bacterial infections.
In search of this information, articles were selected, including 963 primary studies.Of these, 56 were excluded owing to duplication, 482 were not fully available, 377 were excluded after reading the title and abstract, and 17 did not answer the guiding question.Te fnal analysis included 31 articles that were published between 2002 and 2020.Te selection of the studies is presented in a fowchart (Figure 1), as recommended by the PRISMA group [26].
Te majority of the articles were written in English (n � 30, 93.5%), with only one in Portuguese (n � 1, 6.5%).Tere was a higher number of articles published between the years 2011 and 2020 (n � 26, 83.9%), while fewer relevant articles were published during 2002-2010 (n � 5, 16.1%); thus, there was a progressive increase in studies on the antibacterial activity of P. granatum (Table 1).Most studies were conducted in Asia (n � 21, 67.6%), mainly in India (n � 6, 16.1%) and Iran (n � 4, 23.9%).Te origins of pomegranate are believed to be in the Asian Mediterranean, particularly in parts of Iran and northern India, where it spread to the rest of the world.Hence, research is commonly concentrated in this region because of its long history of pomegranate use in healthcare [38].
Te main bacterial strains used in the studies were methicillin-sensitive S. aureus and methicillin-resistant S. aureus (n � 16, 51.6%), and Streptococcus mutans (n � 11, 35.5%) as Gram-positive bacteria; E. coli (n � 16, 51.6%) and Klebsiella pneumoniae (n � 9, 29.0%) as Gram-negative bacteria; and Mycobacterium smegmatis, Mycobacterium bovis, and Mycobacterium tuberculosis (n � 1, 3.2%) as mycobacteria.Te reasoning for investigating the antibacterial activity of P. granatum against these strains may be related to the frequency with which they are involved in infectious diseases acquired in the community and hospital environment, as some of these species have a high capacity to develop antimicrobial resistance [52].Studies investigating the antibacterial activity of natural products found that traditional medicine is facing great challenges in overcoming antimicrobial resistance to commercial antibiotics; therefore, one of its main goals is to fnd alternative compounds capable of combating or reducing bacterial resistance.
Te commonly used methods for evaluating the antibacterial activity of natural extracts were used in the studies, such as the MIC (n � 22, 71.0%), MBC (n � 12, 38.7%), and inhibition halo (n � 23, 74.2%).Tese assays have been used to determine the antimicrobial activity of extracts of P. granatum and several other plant products.Te broth dilution assay and the agar difusion method are more commonly used because of their low cost, speed, and ease of performance compared to other tests [53].
Regarding the solvent extractor, the selectivity, solubility, cost, and safety should be considered when choosing the optimal solvent.Solvents with a polarity value close to that of the solute exhibit better performance, with alcohols (EtOH and MeOH) being the universal solvents for extraction in phytochemical investigations [55].Te main classes of the identifed phytochemical components (Table 2 and Figures 2 and 3) of P. granatum are phenolic acids (ellagic and gallic acids), favonoids (quercetin and anthocyanins), and hydrolyzable tannins (punicalin and punicalagin) [57], all of which have higher solubility in polar solvents, which explains the choice of using MeOH and EtOH as the main extraction agents.
As traditional drug therapies are gradually losing their efectiveness, new therapies based on natural antimicrobial compounds are emerging as alternative treatments for hospital infections [58].Natural products, such as extracts of the P. granatum fruit and other parts containing a wide range of biomolecules, are commonly used because of their antimicrobial activity.Te antimicrobial properties of plant extracts may difer because of variations in their mode of action and chemical composition.Many factors can infuence the antimicrobial activity, such as plant freshness, pharmacogen, extraction method, solvent, geographic region where the plant is grown, and growing season [59].In this study, the most used plant pharmacogens were the bark International Journal of Microbiology (>50% of the studies) and leaves, followed by extracts of the fowers, fruit, petal, stem, pulp, or peel/pulp.Of these, only one did not indicate the portion used to prepare the extract.
Previous studies have shown that compared to other parts of the pomegranate, the peel contains a higher concentration of phenolic compounds, such as favonoids, phenolic acids, and hydrolyzable tannins as the main compounds [60].Tese compounds vary in their chemical nature and play signifcant roles in the plant's antimicrobial, anti-infammatory, and antioxidant activities [61].Tus, the phenolic content of the pomegranate peel is up to three times greater than that in the pulp, which is discarded as biowaste [62].Owing to this high phenolic content, the food industry has been exploring these residues as sources of natural ingredients with possible applications in new products [63].
Pomegranate possesses pharmacological properties, including antiviral and antimicrobial activities conferred by the high polyphenol concentrations, an efect directly attributed to the presence of bioactive compounds of hydrolyzable tannins [64].Te tannin synthesis pathway is regulated by transcription factors expressed during fruit development.Studies have indicated increased expression of the shikimate dehydrogenase and UDP-glucosyltransferase genes of the shikimate pathway, suggesting increased expression of tannins and diferential expression at rind maturation compared to earlier developmental stages of the fruit [65].
Pomegranate peel is an important source of natural bioactive compounds, such as gallic acid, ellagic acid, punicalagin, catechin, and epicatechin, which are also considered the main bioactive compounds in pomegranate bark [66,67].Several researchers have reported the biological activities (antimicrobial, antioxidant, anticancer, antimutagenic, and anti-infammatory) and functions of pomegranate peel.However, some signifcant factors can interfere with the nutritional quality and phytochemical activities of this species, such as the drying methods used to remove water and reduce chemical reactions.Te main drying methods include sun drying, vacuum drying, freeze drying, and oven drying.Furthermore, lyophilization is a potential method for the extraction and recovery of bioactive compounds and other phytochemicals from natural plant sources; however, despite being the best at maintaining the product quality, it is still expensive compared with other methods [68].
Nevertheless, the extraction of bioactive compounds from plant sources is a signifcant step, and diferent solvent systems (polar and nonpolar) have been used to extract and  International Journal of Microbiology 6 International Journal of Microbiology recover polyphenols from plant materials.Generally, nonpolar and low-polarity solvents are used to extract lipophilic compounds and pigments from plants [69].However, the recovery of these substances depends on the type of solvent used and the extraction procedure, which seek to avoid chemical modifcation of the phenolic compounds.Water, ethanol, methanol, and acetone have been described as the most commonly used solvents [70].However, our review revealed that the solvents most frequently used for extracting bioactive compounds were methanol, ethanol and chloroform, hexane, hydroalcohol, and butanol.
In vitro microbiological tests demonstrated that the peel, pulp, and petal extracts of pomegranate inhibited the growth of the main cariogenic bacteria involved in dental caries.Although these are preliminary data, the authors suggested that pomegranate polyphenolic compounds may represent good adjuvants for the prevention and treatment of dental caries but clinical studies are needed to further evaluate their potential [35,41,43,47,49].International Journal of Microbiology

International Journal of Microbiology
Pomegranate does not exhibit antibacterial activity only against bacteria that cause oral infections, as the literature has also shown evidence of inhibitory activity against pathogens that cause intestinal infections.Bacteria known to be the main causes of food infections were tested using the agar difusion assay.Te P. granatum leaf extract inhibited bacterial growth (9.6 mm) and showed the highest concentration index (ΣFIC) when combined with tetracycline (ΣFIC � 0.37) against a strain of E. coli producing metalloβ-lactamase 1 (NDM-1) [31], whereas the peel extracts inhibited the growth of resistant clinical isolates of E. coli (12-13 mm) [11].
Among urinary tract infections (UTIs) agents, E. coli is considered the most frequently isolated and common uropathogen of community-and hospital-acquired infections in 70-90% of UTIs [72].An analysis of the inhibitory efect of three products, namely, crude pomegranate rind extract (PGRE), punicalagin (PG), and pomegranate powder (PGP), on fagellin gene expression and motility of E. coli uropathogenic strain (UPEC CFT073) was performed.Te results showed that normalized luminescence decreased with increasing concentrations of PGRE, PG, or PGP, whereas a decrease in fiC gene transcription correlated with a decrease in the luminescent signal, suggesting that growth in the presence of the products resulted in reduced fiC expression.Tus, this study revealed that PG, PGP, and PGRE at 10% concentration reduced the normalized luminescence signal to 12%, 30%, and 8% of the control signal, respectively, with the strongest inhibitor of fiC expression being PGRE at 10% concentration, resulting in impaired  10 International Journal of Microbiology bacterial motility and reduction in the spread of infection to the upper urinary tract [73].P. granatum also demonstrated bacteriostatic and bactericidal activities against foodborne strains of pathogenic bacteria, inhibiting the growth of E. coli (14.2 mm), S. aureus (18.5 mm), Bacillus cereus (16.3 mm), Salmonella typhi (9.7 mm), and P. aeruginosa (16.1 mm) [33].Similar results were found regarding the antibacterial activity of the P. granatum bark extract on a strain of E. coli (15-22 mm) as well as other foodborne bacteria, such as Salmonella typhimurium (15-23 mm), Salmonella infantis (13-18 mm), Salmonella enterica (15-28 mm), and Salmonella brunei (13-23 mm) [43].Several studies have evidenced the efects of P. granatum extracts against diferent bacteria.Moreover, among the bacterial species described in the literature, some are resistant to conventional antibiotics.
Te main bacteria capable of developing antibacterial resistance include MRSA and P. aeruginosa.MRSA is a multidrug-resistant bacterium that causes nosocomial and community-acquired infections worldwide, usually causing severe infectious diseases, including pyogenic endocarditis, osteomyelitis, and pyogenic infections of the skin and soft tissues.It has several mechanisms (capsular polysaccharides, surface-associated proteins, toxins, and extracellular enzymes) that increase its virulence [74].Analysis of the antimicrobial potential of methanolic and ethanolic extracts of the bark of three fruits (pomegranate, orange, and banana) against pathogenic strains revealed that the ethanolic extract had higher antimicrobial activity against MRSA and S. aureus [75].Te antibacterial potential of the methanolic extract of pomegranate peel can be attributed to the presence of active compounds, such as 5-hydroxymethylfurfural, punicic acid, gallic acid, and punicalagin [76].Tus, there is evidence that plant extracts having high polyphenol content can break the bacterial plasma membrane and inhibit the action of proteins related to antimicrobial resistance [77].
In another study using ethanolic extracts of pomegranate peel (PPEs), mass spectrometry revealed that the most prevalent compounds were punicalagin, ellagic acid, granatin, and punicalin.Analysis of the antibacterial activity revealed that S. aureus ATCC 25923 showed higher sensitivity with MIC and MBC ranging between 0.8 and 6.4 mg/ mL, while another Gram-positive bacterium, Listeria monocytogenes ATCC 19115, showed lower sensitivity with MIC and MBC of 12.8 mg/mL [78].Te antibacterial capacity of pomegranate peel extracts depended on the solvent used, resulting in variations in the biological efects or diferences in the results, which highlights the importance of selecting an appropriate solvent and extraction method for isolating bioactive compounds.
Infections caused by the opportunistic Gram-negative bacillus P. aeruginosa are mainly owed to bioflm formation, which increases its resistance to conventional antibiotics [79].Te use of isolated plant extracts or their combinations has become a promising alternative for inhibiting bacterial bioflms [80].Te susceptibility of P. aeruginosa isolates to plant extracts (pomegranate, thyme, cinnamon, rosemary, clove, and peppermint) was investigated using the agar difusion method, with the results indicating that the extracts exhibited variations in inhibition hanos (12-26 mm).Te pomegranate extract showed the largest inhibition zone (26 mm) against P. aeruginosa, followed by thyme (20 mm), rosemary (18 mm), cloves (17 mm), ginger (16 mm), and peppermint (12 mm).Te data from the present study revealed that pomegranate and rosemary extracts, when tested alone, were able (91.93 and 90.83%, respectively) to inhibit bioflm formation by adhering bacteria to surfaces and reducing colony units and extrapolymer layers.Tese diferences between the extracts can be attributed to the types of active compounds in them.In addition, the combination of both plant extracts with the antibiotic ceftazidime was more efective in eradicating the P. aeruginosa bioflm, ranging from 97.3 to 99.6% [81].Hacioglu et al. [82] reported that pomegranate fower tea in combination with ampicillin had a synergistic efect (FIC � 0.5) against the clinical isolate E. faecalis.
Te growing importance of natural products and their eventual valorization in traditional medicine are due to the need for new alternatives to combat antimicrobial resistance, which directly interferes with the treatment and healing process of infectious diseases that can lead to more severe conditions, thus increasing the mortality rate.Te mechanism of action of P. granatum in clinical isolates should be investigated to clarify the processes involved in its antibacterial activity, thereby enhancing the safety and efcacy of their use in healthcare.Our fndings highlight the potential use of P. granatum extracts in the pharmaceutical industry to develop antimicrobials.

Conclusions
In conclusion, this review revealed that research on P. granatum has been gaining prominence in recent years because it is considered a useful and natural alternative for the development of antibacterial agents.Although the mechanism by which P. granatum extracts modulate bacterial resistance remains unclear, the results suggest that they can act on Gram-positive and Gram-negative bacteria with great potential against multidrug-resistant strains.In addition, pomegranate bark was the most commonly used pharmacogen, whereas methanol, ethanol, and water were the most frequently used solvents for the extraction of bioactive compounds.Te antimicrobial activity of the methanolic extracts of pomegranate peel could be attributed to the presence of active compounds such as 5-hydroxymethylfurfural, punicic acid, gallic acid, and punicalagin.Moreover, pomegranate extracts with a high polyphenol content were capable of disrupting bacterial plasma membranes and inhibiting the activity of proteins associated with antimicrobial resistance.Further studies should be conducted to clarify the mechanism of action behind this biological activity, including the investigation of isolated substances that may be responsible for the antibacterial potential.As clinical studies are still limited, further research is needed to ensure the safety and quality of therapeutic applications of this natural product.

Figure 1 :
Figure 1: Flowchart for study selection according to PRISMA.

Table 1 :
Description of materials, methods, and results of studies that evaluated the antibacterial activity of P. granatum extracts.