63J Contemp Med Sci | Vol. 2, No. 6, Spring 2016: 63–66 Research Investigation of inhibition efficiency of Punica granatum peel extract against bacteria Alaa Abdul-Hussein Al-Daamya, Ula Bahaaa, Dhuha Alaaddina, Marwa Hassana Introduction Bacteria are microscopic single cell called microbes; They are also found in ocean soil and inside the human gut.1 It may be beneficial or harmless (pathogenic). Pathogenic bacteria not only cause disease to human, animals and plants, but also develop more interest for researchers to study in detail.2 Various bacteria cause different types of diseases such as meningitis caused by Neisseria meningitidis; ear infection and strep throat caused by streptococcal bacteria, and toxic shock syndrome caused by Staphylococcus aureus.3 Multiple researches revealed that Punica granatum has pathogen-resistant. P. granatum is one of the oldest known fruits found in writings and artifacts of many culture and reli- gions. It has been revered as a symbol of health, fertility and eternal life.4 P. granatum yields powerful antioxidant such as flavonoids, proanthocyanidins and phenolic.5 Research shows that various extracts from P. granatum has antifungal activity against Listeria monocytogenes, S. aureus, Escherichia coli and Yersinia enterocolitica.6 The literature also reveals that P. granatum has antifungal activity against derma- tophytic fungi and Trichophyton mentagrophytes.7 Our study aimed to examine the antibacterial activity of extract of P. granatum peel against five types of pathogenic bacteria, four is gram-negative P. aeruginosa, Pseudomonas oryzihabita, Proteus varaplis and Klebsiella pneumonia and one gram-positive S. aureus using acetone, ethanol, methanol, ethyl acetate and water as solvents. Materials and Methods Microorganisms: In this study, we used five pathogenic bac- teria isolated from patient in Al-Zahra Teaching Hospital in Karbala city. These bacteria are P. aeruginosa, P. vulgaris, K. pneumonia and S. aureus. Extraction: P. granatum peel was grinded into powder and was uniformly divided in five beakers weighing 20 g each in a beaker. Subsequently, 100 ml of solvent (acetone, ethanol, methanol, ethyl acetate, distill water) with 70% concentration to each beaker that contain plant powder and labeled this beaker with the name of solvent and were kept in a shaking incubator over night with 150 cycle/min at 37°C. The next day, the extracts in all five beakers were filtered using cotton and gauze. Further, the purified extracts in tubes were centrifuged at 3000 rpm for 5 min. After centrifugation, the supernatant was transferred to glass dishes and allowed them to dry. Later, each dish was scrubbed and stored in a clean container and saved as crude extract. The total weight of the crude was also measured.8 Determination of antibacterial activity of the best solvent: First, Muller Hinton agar medium was prepared and sterilized. It was poured in petri dishes and allowed them to solidify. Three wells with 5 mm diameter in every dish were made by cork borer. From the pre-collected powder, 0.05 g from each container was taken and put them in five tubes. Each tube con- tains extract for certain type of our five solvents. 2 ml of eth- anol 70% was added to each tube and shaked until the powder was dissolved. The final concentration of extract in each tube was equal to 25 mg/ml. In addition to our five tubes, a sixth tube was also added containing 2 ml for control (70% ethanol without any extract). After activation of bacteria in nutrient broth, we cul- tured it in the prepared dishes. In each petri dish, 100 μl of each activation bacteria was spread by sterilized spreader, then 50 μl plant extract in each well was added. In the neg- ative control, 50 μl ethanol 70% was added, 50 μg/ml Genta- mycin was used as a positive control. After culturing, all the dishes were incubated in an incubator for 24 hrs under ISSN 2413-0516 aDepartment of Clinical Laboratory, College of Applied Medical Sciences, University of Karbala, Karbala, Iraq. Correspondence to Alaa Abdul-Hussein Al-Daamy (email: aakm7789@gmail.com). (Submitted: 17 March 2016 – Revised version received: 5 April 2016 – Accepted: 24 May 2016 – Published online: 26 June 2016) Objectives The present study aims to determine the inhibitory efficiency of Punica granatum peel extract and test its effectiveness against pathogenic bacteria. Methods Acetone, ethanol, methanol, ethyl acetate and distilled water were used as solvent to obtain of crude extract of P. granatum peel, which tested to determine the effectiveness against five types of pathogens, such as Pseudomonas aeruginosa, Pseudomonas oryzihabitans, Proteus vulgaris, Klebsiella pneumonia and Staphylococcus aureus under a series of concentrations. It was also used to determine the most efficient concentration of solvent optimization, and then was determined minimum inhibitory concentration (MIC) of the extract more efficient. Results The results of the current study showed that the most efficient extraction solvent is ethyl acetate showed the diameters of inhibition zone are 48.66, 51.5, 41.66, 28.0, and 40.83 mm for the types of bacteria above, respectively. The results showed that the concentration of ethyl acetate was 40% in the optimal inhibition of bacteria, amounting to diameters of inhibition zone at the concentration of 19.83, 27.33, 17.66, 15.16 and 12.33 mm for each of the bacterial species above, respectively. The results also found that MIC is 11 mg/ml of P. aeruginosa and 10 mg/ml of P. varaplis and 9 mg/ml of P. oryzihabitans and 3 mg/ml of K. pneumoniae and S. aureus. Conclusion The most effective composites against pathogenic bacteria from P. granatum peel are using ethyl acetate solvent with the concentration of 40%. Keywords Ethyl acetate, Punica granatum, antibacterial activity, MIC. 64 J Contemp Med Sci | Vol. 2, No. 6, Spring 2016: 63–66 Investigation of inhibition efficiency of Punica granatum peel extract against bacteria Research Alaa Abdul-Hussein et al. 37°C. In the next day, after incubation we measure the diameter of zone of inhibition was measured against bac- teria by solvent.9 The optimal concentration of the best solvent: Depending on the results of the previous experiment, ethyl acetate extract was chosen. Five different concentrations of ethyl acetate (20, 40, 60, 80, 100%) were taken in each beaker. In each beaker, 20 g of P. granatum extract powder was added, and the same process of extraction method was repeated that described above and recorded the results. In the next step, 0.05 g of dry extract in five separate tubes was taken, then added 2 ml of 70% ethanol to each tube to obtain on the final concentration 25 mg/ml. After that, 30 petri dishes with Muller Hinton agar were prepared, and followed the three tests to determine the best concentration of ethyl ace- tate, which has the largest inhibition zone.9 Minimum inhibitory concentration (MIC) of ethyl acetate: In a flask, 50 ml of nutrient broth was poured, and in the other flask, 25 ml of nutrient broth was added with 0.625 g from ethyl acetate 40% dry extract to obtain on 25 mg/ml concentra- tion in this flask from extraction. 26 glass tubes were taken and labelled them with numbers from 0 to 25. On each tube, the extract from P. granatum with ethyl acetate 40% concentration which is equal to the number of the label of the tube was taken. For example: in tube number 25, 2 ml from flask 2 was added, which represents 25 mg/ml concentration. In tube number 24, 1.92 ml from flask 2 and 0.08 ml from flask 1 were added, which represents 24 mg/ml concentration, so that with other tubes, in the tube number 0 put only 2 ml from flask 1 to obtain of 0 mg/ml in this tube. The distribution between flask 1 and 2 in these tubes is according to the law C1 V1 = C2 V2. After that, micro titter plate with 96 wells and numbered the wells from 0 to 25, twice for each type of bacteria (each type of bacteria have 52 wells twice for each number), then added 150 μl from each tube in two wells of the same number Table 1. The percentage of materials extracted from P. granatum peel No. Extraction solvent (100 ml) Origin weight of plant powder (g) Weigh of extract (g) Percentage of extract materials (%) 1 Acetone 20 0.49 2.45 2 Ethanol 20 0.6 3 3 Methanol 20 0.5 2.5 4 Ethyl acetate 20 0.65 3.25 5 Distil Water 20 0.457 2.28 Table 2. Inhibition zone (mm) to extract P. granatum peel against bacteria Bacteria Extraction solvent (70%) LSD 0.05Gentamycin 10 μg/ml Acetone Ethanol Methanol Ethyl acetate Distil water P. aeruginosa 21.66 ± 0.88 20.5 ± 0.5 0.0 ± 0.0 27.0 ± 2.59 48.66 ± 3.21 12.83 ± 0.44 4.40 P. oryzihabitans 20.33 ± 0.88 46.66 ± 4.63 39.16 ± 0.83 39.33 ± 0.66 51.5 ± 0.76 39.16 ± 3.0 5.91 P. vulgaris 12.5 ± 0.28 33.0 ± 2.51 31.5 ± 3.25 38.83 ± 1.33 41.66 ± 1.66 23.0 ± 0.57 4.81 K. pneumonia 17.66 ± 0.33 20.66 ± 1.20 18.66 ± 0.16 19.66 ± 0.88 28.0 ± 2.46 26.0 ± 3.60 4.76 S. aureus 17 ± 0.57 39.33 ± 1.83 24.16 ± 1.83 31.0 ± 2.56 40.83 ± 4.4 31.16 ± 0.6 5.94 The numbers refer to mean ± standard error. on each type of bacteria, Then added 50 μl of each type of bacteria(after dilute it with both media and take from the 3rd dilution comparison with McFarland solution) in all the 52 wells from 0 to 25 concentrations. Later, the plates were cov- ered and incubated in an incubator for 24 hr in 37°C. The plates were examined to see growth or no growth of bacteria to determine the minimal inhibition concentration from extract for each type of bacteria.10 Statistical Analysis: Statistical analysis included random complete design(RCD) with 3 replicates. 0.05 is the level of probability that was used to identify the significant differences. The significant differences between the averages were also tested using the test less significant difference (LSD) at the level of probability of 0.05.11 Results The results show the percentage of materials recoverable from P. granatum when we use the solvents: acetone, ethanol, meth- anol, ethyl acetate and distilled water. The percentages are 2.45, 3, 2.5, 3.25, 2.28%, respectively (Table 1). The higher percentage was noted by ethyl acetate 3.25% and low percentage was noted by distilled water 2.28%. The results show that the extract of P. granatum with ethyl acetate give the most large inhibition zone with significance differences (P < 0.05) in all five type of bacteria P. aeruginosa, P. oryzihabita, P. vulgaris, Klebsila pneumonia and S. aureus (Table 2). The diameters of inhibition zone are 48.66, 51.5, 41.66, 28.0, 40.83 mm, respectively. The results were signifi- cantly different (P < 0.05) comparison with Gentamycin and comparison with others solvent. The extract of P. granatum with ( 20, 40, 60, 80, 100)% of ethyl acetate was examined (Table 3). Larger inhibition zone with significant difference (P < 0.05) against gentamycin from hand and in other hand between ethyl acetate 40% and other concentrations was noted. 65J Contemp Med Sci | Vol. 2, No. 6, Spring 2016: 63–66 Research Investigation of inhibition efficiency of Punica granatum peel extract against bacteriaAlaa Abdul-Hussein et al. The diameter with the concentration of 40% that gives 19.83, 27.33, 17.66, 15.16, 12.33 mm against P. aeruginosa, P. oryzihabitans, P. vulgaris, K. pneumonia and S. aureus, respectively. The less concentration of extract that caused inhibition to bacteria (Table 4). It was noted that the concentration 3 mg/ ml of extraction showed inhibition activity against K. pneu- monia and S. aureus, and the concentration 9 mg/ml inhibited P. oryzihabitans, and the concentration 10 mg/ml inhibited P. vulgaris and concentration 11 mg/ml inhibit P. aeruginosa. From this result, it can be noted that bacteria K. pneumonia is more sensitive than the others because it gave less MIC, and in contrast P. aeruginosa gave large MIC. Discussion Various extracts from P. granatum have antibacterial activity against Listeria monocytogen, S. aureus, E. coli and Yersinia entero- cytogene.6 In other study, P. granatum has antibacterial activity against the dermatophytic fungi Trichophyton mentagrophytes.7 Table 3. Inhibition zone (mm) of extract P. granatum peel against bacteria by using dilutions series of ethyl acetate Bacteria Ethyl acetate ratio (%) LSD 0.05Gentamycin 10 μg/ml 20% 40% 60% 80% 100% P. aeruginosa 21.66 ± 0.88 14.66 ± 4.1 19.83 ± 3.52 18.73 ± 1.13 4.66 ± 0.33 0.0 ± 0.0 4.80 P. oryzihabitans 20.33 ± 0.88 16.83 ± 4.1 27.33 ± 1.30 20.83 ± 1.16 13.5 ± 0.28 0.0 ± 0.0 4.68 P. vulgaris 12.5 ± 0.28 15.0 ± 0.28 18.16 ± 1.74 17.66 ± 1.09 17.66 ± 1.16 9.83 ± 0.33 2.49 K. pneumonia 17.66 ± 0.33 15.83 ± 1.09 17.16 ± 2.74 17.16 ± 1.48 15.16 ± 0.44 13.5 ± 0.28 3.45 S. aureus 17 ± 0.57 18.33 ± 0.6 21.0 ± 0.28 15.0 ± 1.52 12.33 ± 1.83 5.0 ± 0.5 2.66 The numbers refer to mean ± standard error. Table 4. Minimum inhibitory concentration (mm) of ethyl acetate extract P. granatum peel Extract Concentration (mg/ml) Types of bacteria P. aeruginosa P. oryzihabitans P. vulgaris K. pneumonia S. aureus 1 + + + + + 2 + + + + + 3 + + + − − 4 + + + − − 5 + + + − − 6 + + + − − 7 + + + − − 8 + + + − − 9 + − + − − 10 + − − − − 11 − − − − − 12 − − − − − 13 − − − − − 14 − − − − − 15 − − − − − 16 − − − − − 17 − − − − − 18 − − − − − 19 − − − − − 20 − − − − − 21 − − − − − 22 − − − − − 23 − − − − − 24 − − − − − 25 − − − − − + means growth − means not growth. 66 J Contemp Med Sci | Vol. 2, No. 6, Spring 2016: 63–66 Investigation of inhibition efficiency of Punica granatum peel extract against bacteria Research Alaa Abdul-Hussein et al. S. aureus and E. coli but it was found that methanol extract had high inhibitory effect. In one study reported that E. coli is more resistant to all P. granatum extract than other Gram -ve bacteria.17 In our study, the difference between previous studies may be in use of different solvents or type of bacteria and also the composition of plant help us to gain more inhibitory effect that contain flavonoids, proanthocyanidins and phenolic. Conclusion From the results, we concluded that the extract of P. granatum peel by ethyl acetate 40% has high antibacterial activity against the pathogenic bacteria that isolated from patients. Recommendations Other study on P. granatum peel is proposed to purify the material, which owns the effectiveness of anti-bacterial pathogenesis.  Previous studies reveal that P. granatum has antibacterial activity against S. aureus, and its juice also has inhibitory effect to S. epidermidis and K. pneumonia.12,13 Methabe et al14 show that methanol, ethanol, acetone and water extract obtained from P. granatum have antibac- terial activity against S. aureus, Salmonella typhi, E. coli, S. dysenteriae, Vibrio cholera, S. soni, Shigella boydii and Shi- gella flexneri . Other study15 found that the extract from Pnica granatum also showed antibacterial activity against E. coli, Enterobacter cloacae, P. fluorescens, Proteus vulgaris, Alcaligenes faecalis, Serratia marcescens, Enterobacter aerogenes, S. aureus, Micro- coccus luteus, Bacillus cereus, Bacillus subtilis, Micrococcus roseus, Mycobacterium phlei, Bacillus coagulans, Micrococcus rodochrus and Mycobacterium smegmatis with inhibition zone 11–31 mm. Ahmed and Beg16 show that alcohol extract of P. granatum have antibacterial activity against E. coli, S. aureus and Shigella dysenteriae. Also Al-Zoreky6 started to notice that the water extract of P. granatum have no antibacterial activity against References 1. Vidyasagar A. Introduction to bacteria, what are bacteria? Live science contributor. 2015. 2. Mcmahon M. Pathogenic bacteria. Wise Greek, 4 April 2015. 3. Health grades editorial staff. Bacterial disease. Health grades, New York, NY . 2013. 4. Seeram NP, Aviram M, Zhang Y. 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