In Vitro and In Vivo Antibacterial Activity of Punica granatum Peel Ethanol Extract against Salmonella

Punica granatum is commonly used in Korea as a traditional medicine for the treatment of pathogenic bacteria. In this study, we investigated the in vitro and in vivo antimicrobial activity of P. granatum peel EtOH extract (PGPE) against 16 strains of Salmonella. The minimal inhibitory concentrations of PGPE were in the range of 62.5–1000 x03BCg mL−1. In addition, the in vivo antibacterial activity of the PGPE extract was examined in a S. typhimurium infection mouse model. Mice were initially infected with S. typhimurium and then with PGPE. The extract was found to have significant effects on mortality and the numbers of viable S. typhimurium recovered from feces. Although clinical signs and histological damage were rarely observed in the treated mice, the untreated controls showed signs of lethargy and histological damage in the liver and spleen. Taken together, the results of this study indicate that PGPE has the potential to provide an effective treatment for salmonellosis.


Introduction
Salmonella enterica, which are Gram-negative bacterial pathogens capable of infecting humans and animals, cause significant morbidity and mortality worldwide [1]. S. enterica serovar typhimurium is a clinically important intracellular bacterial pathogen that causes food poisoning and gastroenteritis in millions of people worldwide each year [2]. The Centers for Disease Control (CDC) estimates that there are nearly 1.4 million food-borne Salmonella infections annually in the USA [3]. This bacterium infects the intestinal tract and causes systemic infection of various organs such as the liver and spleen [4].
Fluoroquinolones and tetracyclines are the antibiotics most commonly used to treat Salmonella, and until recently most strains were susceptible to these drugs. However, a high incidence of Salmonella strains resistant to commonly prescribed antibiotics has recently been reported in Korea and other countries [5,6], and the increased appearance of antibiotic resistant strains of Salmonella further exacerbates this problem [7]. One major concern to public health has been the global dissemination of S. typhimurium Definitive Type 104, which is commonly resistant to five or more antimicrobial agents [8][9][10][11]. The rise in antibiotic-resistant pathogens has led to the development of new therapeutic agents that are effective against these bacteria. Recently, there has been considerable interest in the use of plant materials as an alternative method to control pathogenic microorganisms [12], and many compounds of plant products have been shown to be specifically targeted against resistant pathogenic bacteria [13].
Punica granatum peel is used to treat infections found in human sexual organs as well as mastitis, acne, folliculitis, pile, allergic dermatitis, tympanitis, scalds, diarrhea, dysentery and as an antioxidant [26]. In addition, it is reported that the extracts of P. granatum have antimicrobial activity against Salmonella [23]. However, to date, no studies regarding the antimicrobial activity of P. granatum peels have been conducted. Therefore, the goal of this study is to evaluate the antimicrobial activity of the EtOH extract of P. granatum peel using various in vitro and in vivo models.  (Table 1). In addition, this study included local isolates of S. enteritidis, S. typhimurium, S. gallinarum and S. paratyphi A, which were provided by the National Veterinary Research and Quarantine Service, Republic of Korea. Bacterial strains were suspended in Mueller Hinton broth (MHB, Difco, USA) and then incubated at 37 • C for 20 h. Mueller Hinton agar (MHA, Difco) was used for the agar diffusion method and minimal inhibitory concentration (MIC). Salmonella typhimurium (JOL 389) was used for in vivo assays in mice.

Extraction of Plant
Material. Punica granatum peel was purchased from an Oriental drug store, Daehak Hanyakkuk (Iksan, Korea), and then authenticated by Dr D.Y. Kwon. A voucher specimen (no. 06-022) was deposited in the Laboratory of Herbalogy, College of Pharmacy, Wonkwang University, Iksan, Korea. Next, the P. granatum peel was airdried in the dark at room temperature and then ground into a powder using a mechanical grinder. Approximately 500 g of the powdered materials were then boiled in 1500 mLof EtOH for 3 h. The solvent was then removed under reduced pressure in a rotary evaporator (N-1000S, EYELA, Japan) and dissolved in water or 50% dimethyl sulfoxide (DMSO, Sigma, USA) prior to use.

High-Performance Liquid Chromatography Analysis. The
High-performance liquid chromatography (HPLC) system consisted of a Shimadzu LC-6A model (Shimadzu, Tokyo, Japan), with a column of ODS-C18 (4.6 × 250 mm, 5 µm) and a detection of SPD-6AV with a sensitivity of 0.04 AUFS and a wavelength of 254 nm. Elution was carried out at a flow rate of 0.8 mL/min under a linear gradient of acetonitrile (solvent A) and H 2 O with 1% formic acid (solvent B) from 5% A to 100% A in 50 min. The P. granatum peel EtOH extract (PGPE) was dissolved in a mixture of methanol and water (6 : 4 v v −1 ), and 20 µlL was injected into the HPLC. The presence of gallic acid and ellagic acid was confirmed by the same retention time of their standards (Sigma Chemical Co, St Louis, USA) ( Figure 1). The obtained chromatogram is shown in Figure 2 [27].

Antimicrobial Resistance
Testing. The resistance of the various Salmonella strains to different antimicrobial agents was determined using the disk-agar method standardized by the Clinical and Laboratory Standards Institute [28]. The quality control strain used was Enterococcus faecalis ATCC 29212.

Disc Diffusion Method.
The antibacterial activities of the isolates on the different extracts were tested using the disk-agar method described by the Clinical and Laboratory Standards Institute Standards and by using a modified agarwell diffusion method [28,29]. Briefly, sterile paper discs (6 mm; Toyo Roshi Kaihsa, Japan) were loaded with 20 µL of PGPE (varying concentrations: 100, 200 and 500 µg) dissolved in 50% DMSO and then left to dry for 18 h at 37 • C in a sterile room. The bacterial suspensions were then diluted to a turbidity of approximately 0.5 McFarland (∼1.5 × 10 8 CFU mL −1 ), and then further diluted to obtain the final inoculum. Next, the MHA was poured into Petri dishes and inoculated with 100 µlL of the suspension containing 1 × 10 5 CFU mL −1 of bacteria. Ampicillin (Sigma Chemical Co) was used as the positive control and discs treated with 50% DMSO were used as the negative control. The plates were then placed in an incubator (Vision Co, Seoul, Korea) at 37 • C for 24 h, after which the diameter of the zone of inhibition around each of the discs was measured and recorded. Each experiment was performed in triplicate.

Determination of MICs.
The MIC values were determined for microorganisms that were found to be sensitive to PGPE during the disc diffusion assay. To accomplish this, the microorganism inocula were prepared from 12h broth cultures and the suspensions were then adjusted to a turbidity of 0.5 McFarland. Susceptibility tests were then conducted using the standard broth micro dilution method in accordance with the CLSI guidelines [30] in MHB with an inoculum of ∼5 × 10 4 CFU mL −1 . The MHB was then supplemented with serial dilutions of P. granatum peel of EtOH extracts ranging from 3.9 to 2000 µg mL −1 and ampicillin concentration was ranging from 0.03 to 250 µg mL −1 . The lowest concentration of PGPE capable of inhibiting visible growth after 24 h of incubation at 37 • C was then recorded as the MIC [30].  2.8. In Vivo Assay Using Mice. Mice were divided into the following groups: control (CON), Salmonella-infected (SI) and Salmonella-infected + PGPE (SIPG). Each treatment group contained five mice. Throughout the experiment, mice were provided with water that contained streptomycin (5 mg mL −1 ) in order to reduce the level of facultative anaerobic bacteria that normally colonize the mouse intestine [31]. The inhibition of the growth of test organisms in mice was then determined by monitoring S. typhimurium in the feces of the mice. Briefly, S. typhimurium (JOL 389) was grown overnight in Luria-Bertani broth (Difco), centrifuged, washed in phosphate-buffered saline (PBS) and then diluted into 20% sucrose to achieve a final concentration of 1 × 10 5 CFU. The SI and SIPG groups exclusively were then inoculated using gavage needle orally with approximately 10 5 CFU of S. typhimurium in a 0.1 mL volume. One hour after infection, animals in the SIPG group were orally administered 5 mg (using gavage needle) of the PGPE daily, whereas CON and SI animals were not. Fecal samples were then collected 0, 1, 2, 3, 4, 5 and 6 days after the bacterial suspensions were administered and the numbers of the bacteria per gram of feces were determined. Aliquots (100 µl) of fecal suspensions were serially diluted in PBS and then plated on duplicate Salmonella-Shigella agar plates (Difco), which were subsequently incubated overnight at 37 • C. Typical colonies were then counted on plates that contained between 30 and 300 colonies [32], after which confirmation of S. typhimurium was performed by a PCR assay using a previously described method [33]. At Day 4 post-infection, the mice were sacrificed, and tissue specimens of the kidney, liver, intestine and spleen organs were transferred to 10% buffered neutral formalin for histopathologic examinations and then processed using standard procedures. Sections of paraffin-embedded tissues were then stained with hematoxylin and eosin.

Determination of Antibacterial Activity by the Disc Diffusion Method.
The antimicrobial efficacy of PGPE against the 16 Salmonella strains was evaluated by the disc diffusion method via determination of the surrounding zones of inhibition, as well as by evaluating the MIC using the agar dilution method. Table 2 shows the antimicrobial activity of P. granatum peel extract as determined by the disc diffusion method. The mean values of the zones of inhibition produced against the tested bacteria ranged from 13.3 to 18.6 mm, with the growth of each of the tested strains being inhibited at 500 µg per disc and the zone of inhibition increasing in a dose-dependant manner.

Determination of MICs.
The MICs of the PGPE against the 16 strains of Salmonella are shown in Table 3. The MICs determined using the broth dilution method confirmed the results obtained using the disc diffusion method. PGPE showed antimicrobial activity against each of the tested strains, and these values ranged from 62.5 to 1000 µg mL −1 . The in vivo experiment was therefore conducted with the EtOH extract.

Antibacterial Efficacy of PGPE in Mice.
The in vivo antibacterial activity of PGPE was examined using a mouse S. typhimurium infection model. Briefly, mice were infected with 1 × 10 5 CFU of S. typhimurium (SI). One-hour later,  the mice were orally administered PGPE (SIPG). As shown in Table 4, treatment with the extract of P. granatum peel was found to have marked effects on mortality and on the number of viable S. typhimurium recovered from feces. At Day 1 post-infection, ten mice in the SI and SIPG group shed viable S. typhimurium in feces, with the feces of mice in the SI group being found to contain bacteria at a concentration of 3 × 10 3 to 4 × 10 5 CFU g −1 and feces of mice in the SIPG group being found to contain bacteria at a concentration of 2 × 10 2 to 2 × 10 3 CFU g −1 . In addition, at Day 6 postinjection, none of the mice in the SIPG group had died, whereas all five mice in the SI group had succumbed as illustrated in Figure 3.

Organ Histopathologic
Changes. Salmonella typhimurium-infected mice that did not receive the PGPE were lethargic and showed signs of histological damage in the liver and spleen. In addition, the central and portal veins of the liver showed congestion with focal necrotic emboli-like materials (Figure 4). There were multiple small necrotizing nodular lesions in the liver parenchyma with Kuffer cell hyperplasia and inflammatory cell infiltrate. The spleen showed extensive hemorrhagic necrosis in the red pulp with multiple apoptotic bodies in the white pulp ( Figure 4). No specific abnormal findings were observed in the kidney or the small intestine. Conversely, clinical signs and histological damage were rarely observed in S. typhimurium-infected mice fed with the PGPE.

Discussion
Recently, a number of antibiotics have lost their effectiveness due to the development of resistant strains of bacteria, which has primarily occurred through the expression of resistance genes [34,35]. In addition to inducing resistance, antibiotics are sometimes associated with opposing effects such as hypersensitivity, immune-suppression and allergic reactions [36]. Therefore, there is a need to develop alternative antimicrobial drugs for the treatment of infectious diseases [37,38].
In the present study, the PGPE exhibited antibacterial activity against all 16 strains of eight different Salmonella serotypes tested. In addition, the results of the MIC assays also confirmed its antibacterial effects against all tested salmonella strains. The PGPE also exhibited antibacterial activities against Salmonella strains JOL 389, JOL 411, JOL 419, JOL 420, JOL 421 and JOL 423, all of which have been shown to be resistant to two to five antibiotics ( Table 1). The in vivo antibacterial assay also revealed that the extract effectively inhibited the growth of S. typhimurium and significantly reduced mouse mortality (Table 4). Furthermore, clinical signs of infection and histological damage were rarely observed in test mice, whereas untreated SI mice showed severe clinical signs and histological damage in the tested organs. This is the first study describing the antibacterial activity of P. granatum peel extract against Salmonella. Based on these promising in vitro and in vivo assay findings, we believe that P. granatum peel extract is likely to become a novel antimicrobial treatment for salmonellosis. It has been reported that P. granatum peel extracts have shown antibacterial activity against Escherichia coli O157 and methicillin-resistant Staphylococcus aureus bacteria [14,39]. This antibacterial activity may be indicative of the presence of several metabolic toxins or broad-spectrum antibiotics. Several metabolites from herb species, including alkaloids, tannins and sterols, have previously been associated with antimicrobial activity [40].
In order to investigate components from the PGPE, the HPLC analysis was performed as shown in Figures 2(a) and 2(b). This HPLC analysis among some other minor constituents mainly shows some major phenolic compounds [26]; gallic acid and ellagic acids in addition to punicalagin as a major ellagitannin. The retention time shows it to be gallic acid and ellagic acid [41,42] and also the presence of punicalagin isomers (Figure 1) could be deduced to be one of the major components from the results of literature reported previously [27]. Gallic acid was reported to have antibacterial activity against some intestinal bacteria [43], ellagic acid has anti-microbial activity [44] and punicalagin was reported to show anti-food-borne pathogens [45]. The site and the number of hydroxyl groups on the phenol components may increase the toxicity against the microorganisms [46]. However, it has been reported that gallic acid, ellagic acid and punicalagin have weak antibacterial activity against Salmonella.
The antibacterial activity of P. granatum peel extract might be related to the action of its antibiotic compounds or to the presence of metabolic toxins. This suggests that these components may also provide antibacterial activity against Salmonella and provide a plausible explanation for the higher antibacterial activity of the EtOH extract. On the other hand, the unknown minor components present have not been elucidated in terms of their activity. Further studies then need to be done. In the future, thorough investigation is needed to better ascertain the antibacterial effect of this herb extract.

Funding
Grant No. RTI 05-03-02 from the Regional Technology Innovation Program of the Ministry of Commerce, Industry and Energy (MOCIE) in Republic of Korea.