Study on antibacterial and flavonoid content of ethanolic extract of Punica granatum ( pomegranate ) peel

Pomegranate is known for it’s many health benefits. These benefits are due to the biological active compounds which are present in the pulp as well as in the peel of the pomegranate. The active compounds from the peel were obtained using cold percolation method using ethanol as solvent. Antibacterial activity of pomegranate peel extract (PPE) was studied on E.coli, E. coli NCIM 2065, Salmonella typhi, Salmonella paratyphi B, Pseudomonas aeruginosa, Proteus mirabilis, S. aureus, S. aureus NCIM2079, Shigella flexneri, Lactobacillus casei var shirota by agar well diffusion method. All organisms were sensitive to the extract with inhibitory concentration ranging from 25 mg/mL-100 mg/mL except Lactobacillus casei var shirota which was resistant to even 100 mg/mL concentration of PPE. Flavonoid content of the extract was found to be 10 mg quercetin equivalent/g of extract.


Introduction
Pomegranate (Punica granatum) belongs to the Punicaceae family and is a nutrient dense food source rich in phytochemical compounds.Pomegranates are popularly consumed as fresh fruit, beverages, food products (jams and jellies), and extracts wherein they are used as botanical ingredients in herbal medicines and dietary supplements.Several studies reported that phytochemicals have been identified from various parts of the pomegranate tree and from pomegranate fruit: peel, juice, and seeds. 1 Pomegranate polyphenols include flavonoids (flavonols, flavanols, anthocyanins.),condensed tannins (proanthocyanidins), and hydrolyzable tannins (ellagitannins and gallotannins).Other phytochemicals identified from the pomegranate are organic and phenolic acids, sterols and triterpenoids, fatty acids, triglycerides, and alkaloids. 1Other major components of pomegranate juice are ellagic, caffeic, and punicic acids.These phenolic compounds belong to different representative chemical classes with known bioactivities.
Apart from flavonoids and tannins pomegranate also contains anthocyanins.Anthocyanins possess known pharmacological properties and are used by humans for therapeutic purposes. 2Anthocyanins are the water-soluble pigments responsible for the bright red color of pomegranate.Several anthocyanin compounds identified in pomegranate, include pelargonidin-3-glucoside, cyanidin-3-glucoside, delphinidin-3-glucoside, pelargonidin 3,5-diglucoside, cyanidin 3,5-diglucoside and delphinidin 3,5-diglucoside. 3Anthocyanins can be insect attractants in flowers but can also be insecticidal and antimicrobial at the same time.Tannins as well as anthocyanins have significant antiproliferative and proapoptotic effects in several different types of cancer cells in vitro, including colon cancer, prostate cancer, and head and neck cancer. 4eak levels of these bioactives are found in fruit peel then in the pulp.The peels of fruits are discarded thereby increasing solid waste production, even though they are actually rich in polyphenols.Thus, this waste which is mine of nutraceuticals can be harnessed for its fullest potential.The peels of pomegranate contain 249.4 mg/g of phenolic compounds as compared to only 24.4 mg/g phenolic compounds found in the pulp of pomegranate. 5he probiotics are live microorganisms which exert a beneficial effect on the health of the host when they are administered in adequate quantities (FAO/WHO, 2002) 6 The consumption of living or lyophilized cultures of probiotic bacteria improves the immune system action, prevents cancer, atherosclerosis and coronary diseases.The beneficial effect of probiotics on diarrhoea, gastroenteritis, irritable bowel syndrome, inflammatory bowel disease, lactose digestion, infant allergies, hyperlipidaemia, hepatic disease, Helicobacter pylori infections is well proved.7,8 Functional foods are those foods that provide benefits beyond basic nutrition when consumed as part of the regular diet and help in prevention and treatment of illness and disease.9 When the probiotic bacteria and phytochemicals are combined a product can be developed having double benefits packed in a single product for health and wellbeing.
Thus, the present study evaluates effect of ethanolic pomegranate peel extract on Gram positive and Gram negative pathogenic organisms.And it's effect on probiotic bacteria L. casei var shirota.

Materials
Pomegranates were procured from the local market of Mumbai, Maharashtra, India.All the chemicals (viz, Quercetin, Aluminium Chloride, Dimethyl sulphoxide (DMSO) ethanol and methanol) were of analytical grade and were procured from Sigma Aldrich, Mumbai, India.Sterile Muller and Hinton Broth, Sterile MRS broth, Sterile Nutrient Agar were procured from Hi Media, India.

Test organisms
The

Preparation of extract
The arils of pomegranate were removed and the peel along with albedo was used for preparation of peel extract.The peels were carefully washed under running tap water followed by sterile distilled water.These were air dried at (45°C) for two days, pulverized to a fine powder using a sterilized mixer grinder and stored in air-tight bottles.For the purpose of extraction, a 10 g amount of the pulverized peel was soaked in 90 mL of ethanol (96%) for 24 h.After 24 h the extract was filtered through Whatman filter paper No.1 for removal of peel particles and concentrated under vacuum below 40°C using Heidolph, VE-11 rotaevaporator.The dried extract thus obtained was exposed to UV rays for 2 h and checked for sterility on nutrient agar plates and stored in labelled sterile bottles at 4°C until further use.

Standardisation of inoculum
Bacterial strains were maintained on Sterile Nutrient agar slopes.Bacterial strains were first grown on Mueller Hinton Medium for 18 to 24 h at 37°C.The inoculums of the indicated bacterial strains were transferred into physiological suspension medium and adjusted to 0.5 Mac Farland turbidity standard (10 8 cfu/mL).
The strain of Lactobacillus casei var shirota was isolated on Sterile MRS agar from commercially available probiotic drink Yakult.Incubated at 30°C under anaerobic condition.The purified culture was preserved on sterile MRS agar slants.The inocula of L.casei var shirota was prepared in the similar way as described above.

Antibacterial assay
Antibacterial assay was carried out by using agar well diffusion method. 10Sterile molten Mueller and Hinton at around 40°C was taken and seeded with different micro-bial cultures and plates were prepared.After solidification 6 mm wells were punched.In these wells peel extract dissolved in 10% DMSO at different dilutions were added (100 mg/mL, 50 mg/mL, 25 mg/mL).The plates were incubated overnight at 37°C.After incubation, the zones of inhibition were measured and recorded.Controls were also simultaneously performed.All tests were carried out in triplicates.

Determination of total flavonoid content
Total flavonoid content was estimated by Aluminium chloride colorimetric method. 10,11The principle involved in Aluminium chloride (AlCl 3 ) colorimetric method is that AlCl 3 forms acid stable complexes with the C-4 keto groups and either the C-3 or C-5 hydroxyl group of flavones and flavonols.In addition, it also forms acid labile complexes with the orthodihydroxyl groups in the A-or B-ring of flavonoids.Briefly, 0.5 mL solution of diluted pomegranate peel extract in methanol was separately mixed with 1.5 mL of methanol, 0.1 mL of 10% Aluminium Chloride, 0.1 mL of 1 M potassium acetate, and 2.8 mL of distilled water, and left at room temperature for 30 minutes.The absorbance of the reaction mixture was measured at 415 nm with a double beam Systronic UV/Visible spectrophotometer.Total flavonoid content was calculated as quercetin from a calibration curve.The calibration curve was prepared by preparing quercetin solutions at concentrations 12.5 to 100 mcg/mL in methanol. 12

Statistical analysis
The data obtained were analyzed using SPSS (Statistical Package for Social Sciences) version 11.5 (SPSS Inc., Chicago, Illinois, USA).Descriptive statistics (Mean value and SD) along with comparison in mean zone of inhibition between the extract at different concentrations on different organisms were performed using One Way Analysis of Variance (ANOVA).Confidence level and level of significance were set at 95% and 5% respectively.

Antibacterial activity of extract
In the present study, a good antibacterial activity of ethanolic extract of pomegranate peel was observed.Zone of inhibition of the extract (in mm) significantly increased (p≤0.05) as the concentration of the extract increased.Of the ten organisms used in the study nine were inhibited by the PPE; at concentration range 100-25 mg/mL.Highest Zone of inhibition were seen in given order Shigella flexineri >Salmonella typhi >Staphylococcus aureus >Salmonella typhi paraB ≥Pseudomonas aeruginosa >Proteus mirabilis ≥Staphylococcus aureus NCIM2079 >Escherichia coli >Escherichia coli NCIM2065.While Lactobacillus casei var shirota was resistant to the extract.The Mean zone of inhibition (mm) of ethanol extract of pomegranate (Punica granatum) peel are given in (Table 1).The results are in agreement with earlier investigations reported by other authors to inhibit different microorganisms.Effect of PPE on Bacillus subtilis, Staphylococcus aureus, Escherichia coli, and Salmonella typhimurium has been reported.The MIC for ethanol extract of peels was 242-500 mg/mL against all test bacteria. 13Al-Zoreky found that methanolic extract of pomegranate fruit peels is a potent inhibitor for Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Yersinia enterocolitica. 14In another study by (Sadeghian et al,2011)   fruit skin showed antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. 15In the study carried by Sangeetha et al. antimicrobial activity of Ganesh and Kabul varieties of pomegranate peel extract were evaluated.In the study, they found that Kabul variety the ethyl acetate extract was effective against Bacillus, E.coli, Pseudomonas, Staphylococcus and Candida.The Aqueous extract was effective against Aeromonas, Bacillus, E.coli, Pseudomonas, Staphylococcus, Vibrio and Candida.The methanolic extract of Ganesh variety of Punica granatum showed increased antimicrobial activity against each of the tested strains, and the values ranged from 31.75 to 1000 mcg/mL and in case of Kabul variety the ethyl acetate extract showed activity varying from 62.5 to 500 mcg/mL for selected strains. 16In the work carried by Malviya et al. showed antibacterial activity of ethanol peel extract of pomegranate of Ganesh variety on Staphylococcus aureus, Enterobacter aerogenes, Salmonella typhi and Klebsiella pneumoniae but concentration at which they are inhibited is not mentioned. 17omegranate peel polyphenols, especially tannins are the major components in the pomegranate peel extract that have been implicated in antimicrobial potential i.e. antiviral, antifungal and antibacterial activities. 3In the study by Vasconcelos antibacterial activity of methanolic peel extracts of pomegranate cultivars against both Gram negative and positive bacteria strains have been reported.The MIC values were found to be ranging from 0.25 to 4.0 mg/mL.The author has reported a two-fold MIC value against Staphylococcus aureus than against Escherichia coli. 18It has been suggested that the antimicrobial activity of tannins may be due to the ability of tannin compounds to precipitate proteins, therefore causing leakage of cell membrane of the microorganism , and aiding cell lysis which ultimately leads to cell death. 19As reported by Olaniyi et al.Methanolic pomegranate peel extract showed strong broad-spectrum activity against Gram-positive and Gramnegative bacteria, with the minimum inhibitory concentrations (MIC) ranging from 0.2 to 0.78 mg/mL. 20s documented in the earlier studies the effect of methanol peel extract is done by major researchers.With little work on ethanol extract, here we are reporting that even ethanol pomegranate peel extract is able to inhibit both Gram positive and Gram negative bacteria.

Effect on Lactobacillus casei var shirota
Lactobacillus casei var shirota showed resistance to the PPE at concentration of 100 mg/mL.Bialonska et al. has reported; effect of pomegranate tannin constituents on the growth of various species of human gut bacteria in vitro.The pomegranate by products and punicalagins inhibited the growth of pathogenic Clostridia and Staphyloccocus aureus.Probiotic Lactobacilli and Bifidobacteria were generally not affected by ellagitannins, while a relatively small growth inhibition by ellagic acid likely resulted from decreasing media quality due to the formation of tannin-protein complexes. 21In an investigation found that intake of flavonol-rich foods can modify the composition of the gut microbiota, exerting prebiotic-like effects. 22nabsorbed dietary phenolics and their metabolites have been shown to exert antimicrobial or bacteriostatic activities.These metabolites selectively inhibit pathogen growth and stimulate the growth of commensal bacteria, including also some recognized probiotics thus influencing the microbiota composition. 23

Flavonoid content
The flavonoid content of the pomegranate peel extract was found to be 10mg quercetin equivalent/g of extract.Flavonoids are known to show antioxidant activity having considerable effects on human nutrition and health.The mechanism of flavonoid action is based on scavenging or chelating process. 24

Conclusions
From the study undertaken it can be concluded that ethanolic extract of pomegranate peel has antibacterial properties and good flavonoid content.These properties can be used to formulate new products to be used in food industry as natural antioxidant, replacing synthetic antioxidants, and also as natural food preservatives.Also, resistance of Lactobacillus casei var shirota to PPE makes it a good candidate to be used as an ingredient in preparation of functional foods.
cultures from Culture Collection of Department of Microbiology, Faculty of Science, Jai Hind College, Mumbai were used in the study.The organisms used comprise of six Gram-negative organisms (Escherichia coli, Salmonella typhi, Salmonella paratyphi B, Shigella flexneri, Proteus mirabilis, Pseudomonas aeruginosa) and two Gram positive organisms (Staphylococcus aureus, Lactobacillus casei var shirota).N o n -c o m m e r c i a l u s e o n l y

Table 1 . Mean zone of inhibition (mm) of ethanol extract of Pomegranate (Punica granatum) peel.
aqueous and methanolic extracts of pomegranate R, Resistance.