Portrayal of Punica granatum L. peel extract through High Performance Liquid Chromatography and antimicrobial activity evaluation

Abstract Increasing trend in antimicrobial resistance and failure of chemically synthesized antibiotics lead to discover alternative methods for the treatment of bacterial infections. Various medicinal plants are in use traditionally and their active compounds can be further applied for treatment of bacterial diseases. This study was designed to determine the antibacterial activity of Punica granatum (P. granatum L.) (pomegranate) peel extract against Enterobacteriaceae [Escherichia coli (E. coli), Salmonella Typhimurium (S. Typhimurium) and Shigella Dysenteriae (S. Dysenteriae)] and gram-positive bacterium [Staphylococcus aureus (Staph aureus)]. Methanolic extract of P. granatum L. peel was prepared by Soxhlet apparatus method. Total flavonoid and phenolic contents from the extract were determined by High Performance Liquid Chromatography (HPLC). The antibacterial activity of P. granatum L. peel extract was evaluated through agar well diffusion method. HPLC showed the range of phenolics (gallic acid, caffeic acid, benzoic acid, cinnamic acid) and flavonoid compounds. The chemical structures of flavonoid and phenolics found in the methanolic extract of P. granatum L. peel have been reported for the first time. The methanolic peel extract (50 ul) of yellow P. granatum L. showed 26, 10, 10 and 9mm zones of inhibition (ZOI) against S. aureus, S. Typhimurium, S. Dysenteriae and E. coli, respectively. The methanolic extract of red P. granatum L. (100 ul) showed 27, 8, 12 and 15 mm ZOI against Staph. aureus, S. Typhimurium, S. Dysenteriae and E. coli, respectively. Highest ZOI was observed against Staph. aureus. Many of the bacteria studied in the present work may cause serious gastrointestinal infections, which can lead to hemorrhagic diarrhea in children. These infections can be life-threatening to young children and the elderly. There is an incentive to find alternative control measures, such as plant and herbal extracts, especially in lesser-developed countries where traditional antibiotics may not be readily available.


Portrayal of Punica granatum L. peel extract through High
Performance Liquid Chromatography and antimicrobial activity evaluation

Introduction
Excessive use of antibiotics is the leading cause of resistance in pathogenic bacteria mainly in Staph.aureus, E. coli O157, Salmonella and Pseudomonas.In recent years, the identification of food containing nutrients and biologically active components has been gaining immense attention.The active components in plants (phytochemicals) are helpful components against pathogenic microorganisms in replacement of antibiotics (Devi et al., 2011;Ahmad et al., 2020).
Due to bacterial resistance against antibiotics, herbal medicines can be used for the treatment of many diseases.Medicinal plants are natural products for the treatment of diseases without any adverse side effects (Moghaddam et al., 2013;Javed et al., 2021;Majeed et al., 2021;Rafay et al., 2021).
Different plant extracts have been reported for their antimicrobial activities including some of the important appended here i.e., Aframomum melegueta (Doherty et al., 2011), aqueous and hydroalcoholic extracts of Acanthospermum australe (Loefl.)Kuntze against diarrhea-inducing bacteria (Mallmann et al., 2018), antibacterial and antiproliferative activities of the fresh leaf essential oil of Psidium guajava L. (Silva et al., 2019) and antimicrobial activity of Campomanesia aurea against three strains of Listeria monocytogenes (Pacheco et al., 2020).
Punica granatum (P.granatum L.) locally named as pomegranate is a plant fruit that belongs to the punicaceae (family).It is an important horticultural fruit crop.Polyphenols, important minerals and saccharides are present in edible part of fruit.Various types of phytochemicals are present in the P. granatum L. fruit, tree and seed.The polyphenols are the major class of phytochemicals.Polyphenols have multiple hydroxyl groups on the phenolic rings.Polyphenols have hydrolysable tannins, condensed tannins and flavonoids.The hydrolysable tannin includes ellagitannins and gallotannins (Elalamy et al., 2020).The condensed tannins include pro-anthocyanidins.Flavonols, anthocyanin and flavanols are included in the flavonoids (Abbas et al., 2020).
Brazilian Journal of Biology, 2023, vol.83, e244435 3/7 Antimicrobial use of Pomegranate peel extract range of gram negative and gram-positive bacteria.The active compounds, present in P. granatum L. and their antibacterial activity was evaluated through HPLC and agar well diffusion method, respectively.

Plant sample
The fresh P. granatum L. plant fruits were purchased from the local market of Faisalabad, Punjab, Pakistan.The fruit was confirmed and verified from a botanist following the Bessey classification system.These collected fruit peels were dried and stored safely for further proceedings.

Test organisms
The test organisms were obtained from the culture bank of the Institute of Microbiology, University of Agriculture, Faisalabad Pakistan.The confirmed isolates of three-gram negative bacteria belong to the Enterobacteriaceae E. coli, S. Typhimurium and S. Dysenteriae and a gram positive bacteria Staph.aureus in a final culture strength of 0.5 (1500 × 10 7 cells /ml) were used.MacFarland standard inoculum was prepared in nutrient broth.

Preparation of the P. granatum L. extract
The 10 grams of dried peels of P. granatum L. were crushed in the electric blender machine to make fine powder.Methanolic extract of P. granatum L. peel was prepared through Soxhlet apparatus method in 500ml of methanol solvent by following the already reported method (Lin et al., 2021).P. granatum L. powder was poured in the thimbles and plugged with cotton.The thimble and solvent (methanol) was placed in the Soxhlet apparatus.The extract was prepared after 8 hours of cyclic procedure of the apparatus.The cycles were repeated in the apparatus continuously until the solvent changed to colorless from colored.The extract was removed from apparatus and dried in the rotary evaporator.The extract was stored in the refrigerator at 4°C in the Falcon tubes.

Phytochemical analysis
High Performance Liquid Chromatography (HPLC) was performed for the phytochemical analysis of yellow and red pomegranate ethanolic extracts in Shim-Pack CLC-ODS (C-18) column, 25cm×4.6mm,5µm in the mobile phase of Gradient: A(H2O:AA-94:6.pH=2.27),B(ACN 100%), flow rate was 1ml/min.The detection was done through UV-Vis Detector 280 nm SPD-10Av.Reverse Phase Gradient HPLC made by Shimadzu, Japan was used for analysis.The pump used in this system was LC-10AT.The diluted extracted solution is injected into the HPLC system in a specific volume of 10 µm (Yasmin et al., 2020).

Antibacterial assay
The antibacterial activity of P. granatum L.) peel extract was determined against three gram negative bacteria (E.coli, S. Typhimurium and S. Dysenteriae) and a gram positive bacteria Staph.aureus by following the agar well diffusion method used by Dahiya and Purkayastha, 2012 with little modifications.50µl and 100µl of yellow and red pomegranate ethanolic extracts were tested with the positive (Ampicillin and piperacillin tazobactam) and negative control.Zones were measured after incubation at 37°C for 24 hours.

Minimum Inhibitory Concentration (MIC)
The MIC of P. granatum L. peel extract was determined by broth dilution method.Total 2ml volume contains nutrient broth, inoculum standardized through 0.5 MacFarland standard (1500×10 7 cells /ml).Each tube was incubated at 37°C for 24 hours and results were analyzed.

Antibacterial activity
Chemical structures of flavonoid and phenolics found in the methanolic extract of Punica granatum L. peel is shown in Figure 1.P. granatum L. peel extracts (red and yellow) showed significant antibacterial activity against Staph.aureus, S. Typhimurium, S. Dysenteriae and E. coli.The extract produced greatest zones of inhibition against Staph.aureus as compared to bacteria including even positive control.The zones of inhibition (ZOI) of both red and yellow P. granatum L. extracts against different bacteria have been shown in Table 1 and Figure 2.

Phytochemical analysis of P. granatum L. peel methanol extract
The HPLC chromatogram identified various phenolics and flavonoid compounds in methanolic extract of P. granatum L. peel.The most important phenolic compounds identified through HPLC in both red and yellow pomegranate are given in Table 2.

Discussion
Red and yellow P. grantum L. are already reported and showed comprehensively good results against different microbes.ZOI against E. coli and Staph.aureus determined by Khan and Hanee, 2011 were 25.5 and 22.5 mm, respectively.In current study, a significant difference was observed against Staph.aureus, between methanolic extract of both red and yellow P. granatum L. showed upto 30 mm ZOI in triplicates.It was more than positive control.So, it can be stated that methanolic extract of P. granatum L. peel can be effective against infections that are caused by Staph.aureus and it was more effective against gram positive bacteria than gram negative.In the study carried by Jaisinghani et al., 2018.The inhibition zone was greater against S. Typhimurium than Staph.aureus and E. coli.The zones of inhibition against S. Typhimurium were around 28mm while against Staph.aureus and E. coli were 26 and 23mm, respectively.In current study, the highest zones of inhibition were against Staph.aureus.
Antibacterial activity of P. granatum L. peel extract in ethanol solvent against S. typhimurium showed minimum-inhibitory concentration (MIC) ranged from 62.5 to 1000x03BCgmL−1.The zones of inhibitions were 11, 14 and 15mm against S. Typhimurium.All the three strains showed inhibition zones (Choi et al., 2011).In the current study, the average of zone of inhibition is 16 and 11 mm.There is much similarity in the diameter of inhibition zones against S. Typhimurium.Chebaibi and Filali (2013) reported that antimicrobial activity of P. granatum L. is due to the presence of active components.They isolated the components (phenols, tannins, flavonoids and steroids) but they did not explain the quantities of the active components.We isolated the   same active components and with their quantity in ppm (parts per million).P. granatum L. extract has no antibacterial effects against gram negative bacteria (Kanatt et al., 2010).They reported that there were no effects of P. granatum L. extract on E. coli and S. Typhimurium.In the gram-positive bacteria, P. granatum L. extract showed good antimicrobial activity even at the concentration of 0.01%.In the current study, the P. granatum L. extract shows good antimicrobial activity against both the gram positive and gram-negative bacteria.
P. granatum L. is good antimicrobial agent against gram positive and gram-negative bacteria (Opara et al., 2009).There is much similarity in our results of anti-microbial activity against the pathogenic bacteria (gram positive and gram negative).
Pomegranate has exhibited bactericidal activity against food and waterborne pathogenic bacteria including Salmonella Typhi (S.Typhi) (Perez and Anesini, 1994; Rani and Khullar, 20 04), Vibrio cholerae (Guevara et al., 1994;Mathabe et al., 2006), Yersinia enterocolitica, Shigella spp.(Alanis et al., 2005;Mathabe et al., 2006), and Listeria monocytogenes (L.monocytogenes) (Lucas and Were, 2009;Shan et al., 2011).Typhoid fever (causal agent, S. Typhi) is a life-threatening enteric infection that can be transmitted by consuming food or drinking water contaminated with feces from an infected person particularly pediatric infections.The use of pomegranate extracts as therapeutic intervention is more common in less industrialized countries.Extracts of pomegranate fruit and peel are also tested by agar well diffusion and found to be highly active when compared to a reference concentration-response curve for ampicillin.In one study, ethanolic pomegranate extracts exhibited greater antibacterial activity than the antibiotic chloramphenicol, but lower activity than trimethoprim (Alanis et al., 2005).

Conclusion
From the study undertaken, it can be concluded that methanol extract of P. granatum L. peel has flavonoid and phenolic contents and showed antimicrobial activity particularly against Staph.aureus.Pomegranate extracts may be used therapeutically against public health bacteria and infections causing pediatric infections e.g., diarrhea and dysentery along with several other diseases.

Figure 1 .
Figure 1.Chemical structures of flavonoid and phenolics found in the methanolic extract of Punica granatum L. peel.

Figure 2 .
Figure 2. Zones of inhibition of flavonoid and phenolics found in the methanolic extract of P. granatum L. peel against different public health bacteria.

Table 2 .
Concentration of different flavonoid and phenolics compounds in P. granatum L. methanolic extract determined through HPLC.

Table 1 .
ZOI of bacterial strains against different concentrations of P. grantum L. peel methanolic extracts.