ABSTRACTPomegranate (Punica granatum L.) is rich in polyphenols and other biologically active compounds (anthocyanins, ascorbic acid and І-carotene). Pomegranate extracts (peel, pulp, juice, seed) have antibacterial activity against Escherichia coli (E. coli), Salmonella Typhi (S. typhi) and Shigella dysenteriae (S. dysenteriae) which are gram negative bacteria and Staphylococcus aureus (S. aureus) which is gram positive bacteria. In this study the pomegranate peel and seed extracts will be prepared by vacuum drying oven method and its different dilutions will be tested. Total flavonoid, antioxidant activity and phenolic contents from the extract will be measured with High Performance Liquid Chromatography (HPLC).
The antimicrobial activity of this extract will be checked against the bacteria (E. coli, S. Typhi, S. dysenteriae and S. aureus) by Agar well diffusion method. The proposed study will check the pomegranate peel and seed extract against different bacteria as an antibacterial agent.INTRODUCTIONPomegranate is an ancient fruit that was found in Indus valley. In 1769 the Spanish settlers were introduced it in California.
It previously belongs to punicaceae family but according to recent studies in phylogenetics it belongs to Lythraceae family. The pomegranate fruit is rich source of important saccharides, minerals, and polyphenols. Pomegranate tree (seed, fruit and pulp) has different types of phytochemicals. The polyphenols are the major class of phytochemicals. Polyphenols have multiple hydroxyl groups. The pomegranate polyphenols contains tannins and flavonoids. Proanthocyanidins are present in condensed tannins and hydrolysable tannins includes ellagitannins and gallotannins (Dahham et al., 2010). The ellagitannins significantly accumulates in the large intestine and interacts with complex gut microflora. The pomegranates byproducts inhibits the pathogenic S. aureus and Clostridia. (Bialonska et al., 2009). The extract of P.granatum whole fruit is able to inhibit the production of enterotoxins. (Nuamsetti et al., 2012).The pomegranate juice has the antioxidant activity which can prevent or treat many diseases like rheumatoid arthritis, neurodegenerative diseases, cardiovascular diseases, different types of cancer and skin disorders. Natural pomegranate extract has the advantage of using as an antioxidant in many food and dietary supplements. Pomegranate contains pectin, ascorbic acid and polyphenolic flavonoids. (Fazeli et al., 2011). Pomegranate extracts also have antibiotic properties against those bacterial strains which are developing resistant to conventional antibiotics. The polyphenols of peel extract has a number of activities like they can inhibit microbial growth, oxidation and eliminate free radicles. Pomegranate gel also inhibits the ability of different bacterial strains to adhere to the oral cavity. The pomegranate peel is also used against diarrhea, healing of wound and in the control of bacterial action (Opara et al., 2009). Thus in this study, the methanolic solution of pomegranate peel extract will be prepared and its contents will be quantified by HPLC and the antibacterial activity of this extract will be checked on different bacterial species and their zones of inhibitions will be compared that which bacteria will have the more effect of pomegranate extract. Objectives1. To quantify the phenolic compounds from pomegranate peel.2. To check the antibacterial activity of methanolic extract of pomegranate.REVIEW OF LITERATUREXylia et al. (2018) conducted the study to investigate antibacterial agents of mint and pomegranate extracts against L. monocytogenes and E. coli on shredded carrots. Fresh carrots and pomegranate collected from local market in Limassol, Cyprus and then stored at 4° C until use. Mint plants collected from the experimental farm of Cyprus University. The carrots were dipped into treatment solution (distilled water, mint hydrosol and pomegranate red juice) for few minutes. The results showed the high phenolic contents in pomegranate juice.Jaisinghani et al. (2018) studied antibacterial and flavonoid content of ethanolic extract of pomegranate peel and the purpose was to check its effect on different pathogenic bacteria and on probiotic bacteria L. casei var shirota. Pomegranates were collected from the market of Mumbai, India. The chemicals were taking from Sigma Aldrich, India. The cultures were collected from Culture Collection Department of Microbiology, Faculty of science, Jai Hind College, Mumbai. Total flavonoid contents were measured by Aluminium chloride colorimetric method. Results showed good antimicrobial activity of ethanolic extract of pomegranate peel. Highest zone of inhibition were seen in S. flexineri. L. casei var shirota was resistant to the extract.Naik et al. (2018) conducted the study to assess and compare the antibacterial activity of pomegranate extract with chlorhexidine gluconate (0.2%) against periodontopathic pathogens. The experiment conducted in the Basic Science Research Centre Belagavi. Periodontal pathogens were isolated from subgingival plaque samples which were treated with pomegranate extract (group 1) and with chlorhexidine gluconate (group 2). Results showed that pomegranate extract and chlorhexidine gluconate both were equally effective against the major periodontal pathogens.Setiadhi et al. (2017) investigated acute toxicity of pomegranate seed extract to find its lethal dose values. Experimental research was carried out in The Laboratory of Chemistry Padjadjaran University and The Pharmacology and Laboratory Faculty of Medicine Padjadjaran University, Bandung. Acute systemic toxicity test was performed for ethanolic extract of pomegranate seeds in Swiss Webster mice. They were administered orally with 0.2%, 0.4% and 0.8% per kg. The effects were observed in 30, 60 and 120 minutes. Results showed that there was one mouse dead after the 7th day of test, at 0.8% concentration. Malviya et al. (2014) investigated the most effective solvent to extract the phenolics and antibacterial compounds from pomegranate extract. Pomegranate peel 20g were soaked in six different solvents in 100ml. Results showed the highest yield in ethanol.Moghaddam et al. (2013) studied the effectiveness of pomegranate peel extract against ulcer. Reagents were purchased from Merck, Germany. Pomegranate peel was extracted by soxhlet apparatus with 80% methanol. Peptic ulcer was induced in rats by indomethacin. Test organisms were treated with pomegranate peel extract. The stomachs of rats were dipped in formalin solution to fix the outer layer of stomach. The inhibition was measured. Results showed anti-inflammatory and anti-ulcerogenic effects of pomegranate extract. Qnais et al. (2007) studied the effects of pomegranate aqueous extract against diarrhea. Pomegranates were collected from Amman (Wadi Alseer). Rats were housed in six cages containing six rats each. All rats were administrated by intra peritoneal route. The rats were given 1ml of castor oil and the small intestine was dissected out from them. Water and electrolytes were accumulates in their intestine. They were treated with pomegranate extract. Results showed the reduction in volume and weight of the intestine after the treatment of pomegranate extract. Materials and MethodsSampling Pomegranates will be collected from the market of Faisalabad Pakistan. All the chemicals will be collected from Microbiology Institute, department of veterinary sciences, University Of Agriculture Faisalabad.Test Organisms Three gram negative organisms (E. coli, S. Typhi and S. dysenteriae) and a gram positive organism (S. aureus) tested in the study will be isolated and purified.Positive And Negative Controls: Positive and negative controls will be used in this study.Isolation and Identification Different growth media (MacConkey agar, Blood agar, Nutrient agar) will be used for isolation, identification through colony characteristic, microscopic examination further confirmed through biochemical testing. Preparation of Extract:First of all, pomegranates will be collected and will be washed nicely with distilled water and then it will be used for the preparation of peel extract. Peel will be washed again under tap water and then by distilled water and will be air dried. Peel will be grinded to make fine powder. Powder will be dissolved in methanol and then will be filtered. This extract will be used for further analysis after making its dilution.(Qayyum et al., 2016).High Performance Liquid Chromatography:Extract of pomegranate will be tested through HPLC. The phenolic compounds of pomegranate will be quantified. Peel extract will be injected to the HPLC. (Chidambara et al., 2002).Agar Well Diffusion Method:Antibacterial assay will be checked by agar well diffusion method. The bacterial culture plates (Sterile molten Mueller and Hinton medium) will be used. The prepared peel extract dilutions of pomegranate fruit will be added in the wells and then it will be incubate for 24 hours in the incubator at optimal temperature at 37°C. After incubation the inhibition zones on agar plates will be measured. (Al-Zoreky et al., 2009).Statistical Analysis Data will be analyzed by appropriate statistical method. ReferencesAl-Zoreky NS, 2009. Antimicrobial activity of pomegranate (P. granatum L.) fruit peels. 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