Phytochemical Study and Antibacterial and Antibiotic Modulation Activity of Punica granatum (Pomegranate) Leaves

This study aimed to determine phytochemical contents, antibacterial properties, and antibiotic modulating potential of Punica granatum leaf extracts: hexane, chloroform, ethyl acetate, ethanol, and aqueous extracts as well as an extract enriched with total oligomer flavonoids (TOFs). The TOF extract contained the highest value of phenols and flavonoids. Rutin, luteolin, gallic acid, and ellagic acid were determined by HPLC analysis of this extract. The antibacterial activity was assayed by the disc diffusion method and microdilution method against Staphylococcus aureus and Escherichia coli standard ATCC strains and clinical isolates resistant strains. The TOF extract was the most active against all tested strains. The checkerboard method was used for the determination of synergy between two antibiotics (amoxicillin and cefotaxime) and P. granatum leaf extracts. The best synergistic interaction was found with TOF extract combined with amoxicillin for penicillin-resistant E. coli and penicillin-resistant S. aureus. These results can be assigned to tannins, flavonoids, and phenolic acids found in P. granatum leaf extracts. Pomegranate leaf extracts or active compounds isolated from these extracts could be used to fight the emergence and spread of resistant bacterial strains.


Introduction
According to the 2014 report of the World Health Organization (WHO), antimicrobial resistance is an increasingly serious public health issue that is reaching alarming levels.
e emergence of multidrug-resistant bacteria makes antimicrobial treatments ineffective, lengthens the duration of illness and the time of hospitalization, and increases the mortality rate [1]. One of the solutions is the combination of antibiotics and other agents in order to have a synergistic effect. Many projects aim at developing a new generation of phytopharmaceuticals which can be used alone or in combination with antibiotics. is new generation of phytopharmaceuticals could lend phytotherapy a new legitimacy and it can give the possibility of treating diseases classically treated with synthetic drugs [2].
Punica granatum L. commonly known as pomegranate is a small tree of the family of Lythraceae. It is native from Persia and has been cultivated extensively in the Mediterranean countries such as Tunisia, Turkey, Egypt, and Spain and to some extent also in California, China, Japan, and Russia [3]. Constituents of P. granatum have been involved in many different biological and pharmacological activities. It has been observed that the phenolics contained in pomegranate leaves predominantly contributed to their health benefits. ese phenolics possess a strong binding ability to different molecular structures like proteins or glycoproteins that can antagonize bacterial resistance [2].
Hence, this study aimed to determine phytochemical contents, antibacterial properties, and antibiotic modulation potential of six different pomegranate leaf extracts.

Plant Material.
e leaves of P. granatum (Gabsi cultivar) were collected from the region of Gabes situated in the South of Tunisia, in September 2015. A voucher specimen (Herbarium 23/2015) has been kept in the laboratory of pharmacognosy (Faculty of Pharmacy of Monastir, Tunisia) for future reference. e leaves were shade dried, powdered, and stored in a tightly closed container for further use.

Preparation of Plant Extracts.
Hexane, chloroform, ethyl acetate, and ethanol extracts were obtained by successive Soxhlet extraction (6 h). ese four extracts, with increasing polarities, were concentrated to dryness and kept at 4°C. e aqueous extract was prepared by a decoction of 50 g of plant material in 500 mL of boiling water for 20 minutes. After filtration, the extract was lyophilized and kept at 4°C. e extract enriched with total oligomer flavonoids (TOFs) was obtained, as described by [4], by maceration in a mixture of acetone/water (2 V/1 V). After filtration, the acetone was evaporated under reduced pressure and the residual aqueous phase was saturated with NaCl. e formed precipitate overnight at 4°C was removed by filtration. Liquid-liquid extraction was carried out with ethyl acetate. e organic phase was concentrated under reduced pressure and then poured into an excess of chloroform. e resulting flocculate corresponds to the TOF.
For phytochemical study and antibacterial and antibiotic modulation assays, each extract was dissolved in the adequate solvent. e same solvent was used in the corresponding negative control.

Phytochemical Study
2.3.1. Preliminary Phytochemical Screening. Extracts were screened for the presence of secondary metabolites such as tannins, flavonoids, saponins, cardiac glycosides, anthocyanins, and alkaloids. e secondary metabolites were detected qualitatively by the characteristic color changes occurring upon treatment with specific reagents [5].

Determination of Total Contents of Phenols, Flavonoids, and Tannins.
e total content of phenols of extracts from P. granatum was determined by the Folin-Ciocalteau method [6]. Indeed, to 100 μL of a solution of extract, 2 mL of sodium carbonate (Na 2 CO 3 ) at 2% and 0.1 mL of Folin-Ciocalteu reagent at 50% were added. After incubation for 30 minutes at room temperature, a petroleum blue color indicates the presence of polyphenols which were measured at the wavelength of 720 nm. Gallic acid was used as a standard, and results were expressed as milligram of gallic acid equivalents (GAEs) per gram of dry mass (DM). e content of flavonoids of each extract was determined by the aluminum chloride colorimetric method [7]. is method consists in adding 75 μL of 5% NaNO 2 , 150 μL of 10% AlCl 3 , and finally 500 μL of 1 N NaOH to 250 μL of a solution of leaf extract. e volume was adjusted to 2.5 mL and incubated for 5 minutes at room temperature. Absorbance was measured at 510 nm. Quercetin was used as a standard and results were expressed as milligram of quercetin equivalents (QEs) per gram of DM. e content of tannins was determined by the Folin-Denis [8]. We added 10 μL of Folin-Denis reagent to 50 μL of extract, followed by 25 μL of saturated sodium carbonate solution, which was then incubated at room temperature for 90 minutes. e absorbance was measured at 760 nm. Tannic acid was used as standard and results were expressed as milligram of tannic acid equivalents (TAEs) per 100 g of DM.

HPLC Characterization of TOF Extraction.
A highperformance liquid chromatographic method with gradient elution and diode-array detection was used to quantify some phenolic acids (gallic acid, ellagic acid) and flavonoids (rutin, luteolin) in TOF extract.
HPLC analyses were performed with Shimadzu LC-2030 3D Plus (Prominence-i) chromatograph. e mobile phase contains 1% aqueous acetic acid solution (solvent A) and acetonitrile (solvent B), the flow rate was adjusted to 0.7 mL/ min, the column was thermostatically controlled at 28°C, and the injection volume was kept at 10 μL. Gradient elution was performed by varying the proportion of solvent B to solvent A: from 10% to 40% B for 28 min, from 40 to 60% B for 39 min, and from 60 to 90% B for 50 min. e mobile phase composition was set back to the initial condition (solvent B: solvent A: 10 : 90) in 55 min and allowed to run for another 10 min, before the injection of another sample. Chromatographic separation was performed on a Chrom-CloneTM C18 column (5 μm particle size, 250 × 4.6 mm), and the detection was conducted using a diode-array UV detector at 254 nm [9].
Standard solution of gallic acid, ellagic acid, rutin, and luteolin was prepared in methanol (0.1 mg/mL). e sample solution was prepared by dissolving 1 mg of TOF extract in 1 of mL methanol. Both the standard and sample solutions were filtered through a Whatman 0.45 μm syringe filter. e responses were measured as peak areas versus concentration.

Bacterial Strains.
Antibacterial activity of P. granatum extracts was determined against Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli). Extracts were tested against bacterial strains obtained from the American Type Culture Collection (S. aureus ATCC 25923 and E. coli ATCC 25922) and clinical isolates strains obtained from the department of microbiology (University Hospital of Monastir) with resistance profile described in Table 1.

Disc Diffusion Method.
e disc diffusion method was performed according to the method reported by the National Committee for Clinical Laboratory Standards (NCCLS). Sterile paper disks of 6 mm diameter were impregnated with different extracts and then air-dried. Each disk contains approximately 20 μL of extract at the concentration of 5 mg/mL. Blank disks, impregnated with solvents, were used as a negative control. ese paper discs were placed on Müller-Hinton agar inoculated with the tested bacteria (10 8 CFU/mL). Following incubation at 37°C for 24 hours, diameters of inhibition zones were measured in millimeter scale.

Microdilution Method.
e microdilution method using 96-well microplates and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to indicate bacterial growth was used to determine the Minimal Inhibitory Concentration (MIC) [10,11]. An inoculum of 100 μL of each strain suspended in Müller-Hinton broth up to a final concentration of 10 6 CFU/mL was used in 96-well microplates. Each well received 100 μL of each extract solution. Using twofold serial dilutions, the final concentrations of the extracts varied from 5 to 0.04 mg/mL. After incubation at 37°C for 24 hours, 40 μL of MTT at the concentration of 0.2 mg/mL was added to each well. After incubation for 30 minutes, MICs, which are the lowest concentrations required to inhibit growth, were recorded as the lowest concentrations required to inhibit the color change of the bacterial suspension to blue.

Antibiotic Modulation.
e checkerboard method, which is commonly used for the determination of synergy between the antibiotics and natural antibacterials, was used for the antibiotic modulation assay [12].
is method is combined with a calculation of a fractional inhibitory concentration (FIC) index. To evaluate the synergy of our extracts with amoxicillin and cefotaxime, we started by determining the MIC of these antibiotics (MIC ATB ) and the MIC of our extracts (MIC Ext ) alone, and then we determined their new MIC after combination (MIC' ATB and MIC' Ext ). FIC must be determined to judge the interaction between the antibiotic and the natural extract: FIC � FIC A + FIC B with FIC A � MIC' ATB /MIC ATB and FIC B � MIC' Ext /MIC Ext "Synergy" was defined by an FIC index ≤ 0.5. When the FIC index fell between 0.5 and 4.0, it indicated that there was "no interaction" between the agents. An FIC index � 4.0 would indicate that there was an "antagonism" between the two agents. e microdilution method using tetrazolium salt [10] was used to determine the MIC of plant extracts and MIC of amoxicillin and cefotaxime. e initial concentrations of the agents used were determined according to their MIC value and then were serially diluted in twofold steps.

Results and Discussion
3.1. Phytochemical Study. In this study, six extracts were prepared from the powdered leaves of P. granatum. e yield of extracts depended on extraction solvents and extraction methods and varied from 0.5 to 20.07% (Table 2). e highest extraction yield was obtained with the aqueous extract and the lowest yield was obtained with the ethyl acetate extract.  [13][14][15]. But, some reports found, in addition to flavonoids and tannins, other secondary metabolites such as alkaloids [16] and anthocyanins [17].
Based on the results of the preliminary phytochemical study, the total contents of phenols, flavonoids, and tannins in leaf extracts of P. granatum were determined. ese results are shown in Table 2. e TOF contained the highest value of phenols (382.27 ± 6.46 mg GAE/g DM) and flavonoids (492.88 ± 19.25 mg QE/g DM), followed by the aqueous extract (262.98 ± 5.11 mg GAE/g DM and 287.33 ± 15.55 mg QE/g DM, respectively). e aqueous extract showed the highest content of tannins (125.92 ± 3.9 mg TAE/100 g DM) followed by the ethanol extract (96.81 ± 2.27 mg TAE/100 g DM). Phenols, flavonoids, and tannins were absent in hexane extract. We also did not find flavonoids in chloroform extract. Polar extracts are richer in different secondary metabolites than nonpolar extracts. In fact, the phenolic nature of polyphenols such as flavonoids and tannins makes them relatively hydrophilic and highly soluble in water and polar organic solvents such as methanol, ethanol, and acetonitrile, or their mixture of water [18]. TOF contained the highest value of phenols and flavonoids but contained fewer tannins since it has been obtained using purification methods that removed tannins and enriched the extract with flavonoids. e total contents of phenols (410.85 ± 10.15 mg GAE/g DM) and flavonoids (286.88 ± 20.73 mg QE/g DM) in our study are greater than those reported by [17] which are 289.76 ± 1.55 mg GAE/g DM and 82.6 ± 2.67 QE/g DM, respectively, in extracts obtained by successive extraction using cold maceration method with solvents of increasing polarity (hexane, dichloromethane, ethyl acetate, ethanol, and methanol). Such a difference may be explained by the extraction method and the harvest time. Indeed, we prepared our extracts by successive extraction using the Soxhlet apparatus which gives better efficiency than cold maceration. Soxhlet method uses a fresh solvent that is always provided to the sample causing maximum analyte solubility [19]. Harvest time has an influence on the amount of phenols and flavonoids. Total levels of phenolics and flavonoids in pomegranate leaves are low in the early stages of leaf growth and then increased gradually until the end of September [16]. Regarding tannins assay, we found the highest values in polar extracts (aqueous extract, ethanol extract). Tannins are usually extracted from natural tissues by aqueous solutions of methanol, ethanol, or acetone, as well as ethyl acetate. Nonpolar organic solvents (hexane, chloroform, and dichloromethane) have low extraction strength for tannins [20].

Antibacterial Activity.
e antibacterial activity of P. granatum extracts was assayed in vitro by agar disc diffusion method and microdilution method against Gram-positive and Gram-negative human pathogenic bacteria. Table 3 summarizes the microbial growth inhibition of different extracts. e in vitro antibacterial study against the standard and resistant strains of S. aureus and E. coli showed that P. granatum leaf extracts exhibited various levels of antibacterial effect against all tested bacterial strains. Both techniques, disc diffusion and microdilution, have given consistent results. e inhibition zone diameters varied from 8 to 19 mm and MIC values varied from 0.625 to over 5 mg/ mL. e TOF extract was the most active against all tested strains. In fact, this extract showed a remarkable activity against the Gram-positive and Gram-negative standard strains (MIC � 0.625 mg/mL) and the penicillin-resistant strains (MIC � 2.5 mg/mL). It also displayed an interesting activity against the methicillin-resistant S. aureus (MIC � 1.25 mg/mL) which is a major nosocomial pathogen worldwide and only some antibiotics such as vancomycin and teicoplanin are active against it [22]. e ethanol extract was active against all S. aureus strains and standard E. coli strains (MIC � 2.5 mg/mL). e aqueous extract was especially active against E. coli standard strain (MIC � 1.25 mg/ mL) and S. aureus standard strain (MIC � 2.5 mg/mL).
is antimicrobial activity can be assigned to tannins and flavonoids found in P. granatum leaf extracts. ese phytochemical groups are known to possess antimicrobial compounds [23,24]. eir presence in the plant extracts could, therefore, justify the observed activity especially with the TOF extract, aqueous extract, and the ethanol extracts e antimicrobial effects of pomegranate leaves were previously studied by the disk diffusion method. Indeed, these studies showed that the methanolic extract exhibited the best inhibition against all tested strains (Staphylococcus aureus, Bacillus cereus, Escherichia coli, Proteus mirabilis, and Salmonella typhi) [25]. Methanolic leaf extract of P. granatum also showed antibacterial activity against the predominant bacterial isolates of septic wounds that are multidrug-resistant P. aeruginosa, S. aureus, K. pneumoniae, and E. coli [26]. e antimicrobial activity of ethyl acetate extract of pomegranate leaves was also studied by the microdilution method and showed MIC of 3.75 mg/mL for P. aeruginosa, MIC of 7.5 mg/mL for E. coli, and MIC of 15 mg/mL for S. aureus [27].

Antibiotic Modulation.
e potential reduction of antibiotic resistance by P. granatum extracts was carried out by calculation of the FIC index shown in Table 4. e best synergistic interaction was found with TOF extract combined with amoxicillin for penicillin-resistant E. coli and penicillin-resistant S. aureus (FIC � 0.125). TOF extract showed also synergistic effect with cefotaxime for methicillin-resistant S. aureus (FIC � 0.5). e aqueous extract and ethanol extract were found to have a similar effect in reducing the resistance of penicillin-resistant S. aureus with amoxicillin (FIC � 0.25). Chloroform extract and ethyl acetate extract showed no synergistic effect with amoxicillin and cefotaxime for all tested resistant strains.
In the interaction study, polar extracts such as TOF, ethanol, and aqueous extracts were found to be highly effective especially against penicillin-resistant strains. ese extracts contained the highest amount of phenolic compounds such as flavonoids. In fact, many studies showed that, in synergy with antibiotics, phenolic compounds such as flavonoids, tannins, and phenolic acids pose a promising alternative for therapeutic strategies against drug-resistant bacteria. e proposed mechanisms of synergy were the copermeabilization of the bacterial membrane and the inhibition of penicillinase activity [28,29]. e work [30] showed that pomegranate ethanol extract inhibited recombinant New Delhi metallo-β-lactamase 1 (NDM-1) activity and the IC 50 value was 0.76 ng/μL. e same extract caused an increase in NDM-1 E. coli cell permeability. In addition, methanol P. granatum leaf extract was able to produce a significant inhibition of biofilm formation for L. monocytogenes and S. aureus [31]. It should be noted that the   formation of biofilm is an important mechanism of bacterial resistance. Finally, gallic acid, which is a phenolic acid present in TOF extract, showed a synergistic interaction with tetracycline by inhibiting the efflux pump in multidrugresistant E. coli [32]. To our knowledge, no study in literature measured the antibiotic modulation potential of pomegranate leaf extracts with β-lactam antibiotics.

Conclusion
e phytochemical preliminary screening of P. granatum leaf extracts showed the presence of hydrolysable tannins and flavonoids. e quantitative analysis of total phenolics, flavonoids, and tannins carried out with spectrophotometric methods had confirmed these results particularly with TOF, aqueous, and ethanol extracts.
e antibacterial study showed that the TOF extract was the most active extract against all tested strains. e most important result was the activity shown by the former extract against the methicillinresistant Staphylococcus aureus. TOF extract also showed the best synergistic interaction with amoxicillin for penicillinresistant E. coli and penicillin-resistant S. aureus. HPLC determination of flavonoids and phenolic acids in TOF extract showed the presence of rutin, luteolin, gallic acid, ellagic acid, and other undetermined substances.
In conclusion, pomegranate leaf extracts or active compounds isolated from those extracts could be used to improve human health especially the prevention and treatment of infectious diseases and to fight the emergence and spread of resistant bacterial strains. Nevertheless, further researches are still needed to evaluate the mechanisms of action and toxicity of those extract.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.