In view of the large number of the plant species potentially available for study, it is essential to have efficient systems of the methods to evaluate efficacy of medicinal plants as antimicrobial agent. The evaluation for antimicrobial agent of plant origin begins with thorough biological evaluation of plant extracts to ensure efficacy and safety followed by identification of active principles, dosage formulations, efficacy and pharmacokinetic profile of the new drug .
Punica granatum is a fruit-bearing deciduous shrub or small tree. It belongs to family Punicaceae and distributed throughout the tropics and sub tropics, historically it was believed to be a symbol of fertility. It could be shrub or tree and usually grows to < 5 m, but can occasionally reach 12 m in favorable conditions . The leaves are shiny and about 7.6 cm long . Different parts of the plant such as bark, leaves, immature fruits and fruit rind have medicinal significance . Punica granatum has been extensively used as a traditional medicine in many countries for the treatment of dysentery, diarrhea, helminthiasis, acidosis, hemorrhage and respiratory pathologies . Additionally, this plant is reported to have excellent antibacterial, antifungal, antiprotozoal and antioxidant properties [11-13]. Numerous phytochemical constituents have been reported to be present in different parts of the pomegranate plant making it pharmacologically precious [14,15].
However, the antimicrobial compounds from plants may inhibit growth of microorganisms by different mechanisms than those presently used . So, there is a need to develop plant based new antibiotics which are effective against resistant microorganisms. So that, this research was done to evaluate the potential of plant extracts on clinical isolates of drug resistant bacteria strains and fungus (Candidia albicans). Moreover, synergistic effects of crude extracts in association with pure prescribed antibiotics were investigated against all the test bacteria and fungus.
Material and Methods
The study was conducted at Jig-jiga University in the Department of Biology which is located 675km away from Addis Ababa in Eastern Ethiopia from September, 2014 to April, 2015 and the laboratory work has been accomplished at Addis Ababa University.
Collection of plant materials
Each plant species Punica granatum was collected from Bobas, where the plants are widely available, and identified further in herbarium at Addis Ababa University, Botanical Herbarium. The fresh leaves of each plant was washed three times with pure (sterilized) water, allowed it to air dry at room temperature under shade (exposure to sun light was avoided to prevent loss of active components). The dried leaves were powdered with the help of pestle and mortar and store in sterile bottle  for further analysis.
Extraction of plant leaves
In this study, 50g of shade-dried powder was loaded in 1000 ml flask and mixed with 250 ml of 95% methanol, acetone, chloroform and sterilized water solvents for extraction and then shacked on a rotary shaker for three days. The extracts was filtered by passing through Whatman's filter paper No:1 and centrifuged at 5000 rpm for 15min. The solvent extracts was concentrated under reduced pressure using Rotavapor and preserve at 4 degree C in air tight bottle for further investigation.
Standard and drug resistant pathogenic bacteria were used for determination of antimicrobial activities of different extracts. In brief, the bacterial strains which was used to assess the antimicrobial properties of extracts includes both standard and drug resistant clinical isolates of gram positive and gram negative strains: Staphylococcus aureus (S. aureus) (MTCC2001), Streptococcus pneumonia (S. pneumoniae) (MTCC5954), Escherichia coli (E. coli) (MTCC8650), Shigella boydii (S. boydii) (MTCC7704) and as well as Candida albicans (C. albicans). They were obtained from Pasture Institute of Microbiology and were maintained on Nutrient agar (NA) and Sabouraud-dextrose agar (SDA) (for the fungal strain only) and sub cultured before use.
Preparation of inoculums
The microbial stock cultures were maintained at 4 degree C on slopes of nutrient agar. Active cultures for experiments was prepared by transferring a loopful of cells from the stock cultures to test tubes of Muller-Hinton broth (MHB) and was incubated without agitation for 24 hrs at 37 degree C. To 5 ml of saline solution, 100 μl of culture was inoculated and incubated till it reached the turbidity equal to 0.5 McFarland standard solutions which is equivalent to 106-108 CFU/ml .
Antimicrobial activity assay
The antimicrobial activity assay was done by agar well diffusion method. In short, antibacterial activities of the different extracts were determined by agar well diffusion assay on MHA medium. The MHA was melted and cooled to 48 - 50 degree C and poured into sterile Petri dishes to give a solid plate. Then standardized inoculums of 100 μl (1.5×108 CFU/ml, 0.5 McFarland) was added aseptically and streaked on the agar plate surface. Wells were prepared in the seeded agar plates with sterile cork borer (6 mm diameter). The test compound or crude extract (100 μl) was carefully dispensed into the wells (6 mm). This was done in triplicate in parallel to different control antibiotics. Extracts were allowed to diffuse for about 2 hrs before incubation. Plates were incubated overnight at 37 degree C. After overnight incubation, the plates were observed for the zone of inhibition and the diameter of the inhibition zone was measured. The presence of a zone of inhibition around each well was indicative of antibacterial activity. The diameters of zone of inhibition produced by the extract agents were compared with those produced by the commercial control antibiotics.
Determination of MIC and MBC/MFC
The Minimum Inhibitory Concentration (MIC) of Punica granatum leave extracts were determined according to methods described by . Each extracts were diluted to concentrations ranging from 6.25% to 50%. To each dilution of leave extract, nutrient broth tubes were seeded with 100 μl of the pathogenic standard and resistant clinical bacterial inoculums, and clinical isolate of the fungus. Negative control tubes with no bacterial and fungal inoculation was simultaneously maintained. Tubes were incubated aerobically at 37 degree C for 24 h. The lowest concentration of the extract that produced no visible growth (turbidity) was recorded as the MIC. Dilutions showing no visible growth for the MIC were sub cultured onto a fresh MHA plate and incubated at 37 degree C for 24 h for the determination of Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC). In brief, dilution showing no visible growth in the determination of MIC was streaked (sub cultured) on to MHA and incubated for 24 h for the bacteria and the fungus. The least concentration of the extract with no visible growth after incubation was taken as the MBC and MFC.
Synergetic effect determination of crude plant extracts and pure antibiotics
The crude extracts and standard antibiotics were diluted to (10% chloroform extract, 10%, amoxicillin, 10% vancomycin) as well as combination of extracts with clinically prescribed drugs were diluted to (10% extract plus 10% (amoxicillin, vancomycin) to evaluate whether synergetic effect was there or not. Then 100 μl of each crude extract was added into the wells of 6 mm to examine inhibition zone differences after incubation .
The data was analyzed using SPSS version 21.0. Means and standard deviations of the triplicate analysis were calculated using analysis of variance (ANOVA) to determine the significance differences between the means using Duncan's Multiple range test (p ≤ 0.05) when the F-test demonstrated significance. The statistically significant difference was determined as p ≤ 0.05.
The plants were extracted using four different solvents to increase the possibility of getting the bioactive components, thus their leaf were treated with each solvent chosen and the crude extracts yielded are calculated in percentage. From 50g of Punica granatum leaf powder, 23%, 24%, 14% and 21% of crude antibiotics were collected for diffusion and dilution analysis with acetone, methanol, and chloroform and water solvents, respectively.
Determination of antimicrobial activity of plant extracts
The extracts were tested for the antimicrobial activity against the selected bacterial and fungal strains using agar well diffusion assay and broth dilution methods.
Agar well diffusion method
All extracts of Punica granatum against S. aureus (MTCC2001) showed various degrees of antibacterial activity. Both methanol (22.3 +/- 1.15) and chloroform (19.6 +/- 2.88) crude extracts were resulted significantly (p ≤ 0.05) wider clear zones than the disc antibiotics of methicillin (12.3 +/- 2.30) and amoxicillin (13 +/- 1.00). On the other hand, only methanol (22.3 +/- 1.15) extracts were showed better activity than to that of water (15.0 +/- 2.64) among all sample extracts against on the same strain.
Inhibition zones of methanol (22.6 +/- 5.50) and chloroform (20.3 +/- 7.57) extracts were significantly (p≤ 0.05) greater than the all commercial antibiotics vancomycin (15 +/- .00), methicillin (0 +/- .00) and amoxicillin (11.6 +/- 2.8) towards MRSA. Similarly, acetone (18.0 ± 5.0) and water (12.0 +/- 3.60) inhibition zones were statistically greater than methicillin and amoxicillin positive controls on MRSA. Moreover, there was no statistically significant (p>0.05) inhibition difference observed between the three solvent crude extracts, although greater the water extracts (Table 1).
Among all the crude antibiotics of the same plant tested against S. pneumonia (MTCC5954), acetone (21.6 +/- 1.52) extracts were showed significantly greater clear zones than the controls vancomycin (16.6 +/- 2.51), methicillin (10.3 +/- 1.52), amoxicillin (15.6 +/- 1.15), and to other solvent extracts methanol (12.3 +/- 0.57), water (16.0 +/- .00) and chloroform (14.3 +/- 0.57). Alternatively, methanol, water and chloroform extracts were showed considerable antimicrobial effect with no significant difference between each other (Table 2).
The study was also showed that acetone (17.0 +/- 6.24), water (15.6 +/- 2.30) and chloroform (17.6 +/- 0.57) extracts Punica granatum were measured a significantly (p≤ 0.05) greater inhibition zones than the parallel tested commercial antibiotics vancomycin (10.0 +/- .00), methicillin (0 +/- .00) and amoxicillin (0 +/- .00) towards the drug resistant S. pneumonia, however no significant inhibition zones difference obtained between the solvent extract themselves. Comparably methanol (10.3 +/- 0.57) extracts did not elicit statistically better activity than vancomycin (10.0 +/- .00) control antibiotics on the same resistant pathogen. Values are means of triplicate determination values within the same row and column followed by different superscripts are statistically different (p≤ 0.05) against each tested microorganisms.
Acetone (18.6 +/- 4.6), methanol (17.0 +/- 1.00) and chloroform (15.0 +/- .00) extracts of the same plant were recorded a significantly (p≤ 0.05) greater inhibition zone than vancomycin (12.3 +/- 0.57), methicillin (8.6 +/- 0.57) and amoxicillin (11.0 +/- 1.00) against E. coli (MTCC8650), though no significant clear zone difference obtained among themselves. And there was no antibacterial activity showed with the water (0 +/- .00) extracts. All, except water, extracts of Punica granatum provoke a remarkable antibacterial activity and efficiency with inhibition zones of acetone (14.3 +/- 2.51), methanol (12.0 +/- 3.46) and chloroform (13.0 +/- 1.00) extracts against drug resistant E. coli with no significant difference between one another. Nevertheless, all the controls and water extracts were exhibited no activity at all on the same test bacteria.
Crude extracts of acetone (14.0 +/- .00) Inhibition zone was significantly (p≤ 0.05) greater than vancomycin (11.3 +/- 0.57), methicillin (0+/-.00) and amoxicillin (10.3 +/- 2.08) against S. boydii (MTCC7704). Unlikely, methanol (13.0 +/- 1.00) and chloroform (11.3 +/- 3.21) extracts were showed no significant (p>0.05) clear zone difference with that of the controls vancomycin and methicillin. However, the water extracts of the same plant didn't induce significantly better potential over the controls used on the standard bacteria.
Punica granatum extracts of all crude antibiotics were also showed a various range of activities on C. albicans. Inhibition zones of acetone (15.3 +/- 1.52) and chloroform (16.6 +/- 1.52) were significantly (p≤ 0.05) greater than the other two methanol (10.0 +/- 1.00) and water (11.3 +/- 0.57) solvent extracts, but there was no antifungal activities were shown by the controls (Table 3). Values are means of triplicate determination values within the same row and column followed by different superscripts are statistically different (p≤ 0.05) against each tested microorganisms.
MIC and MBC determination
The crude antibiotics which were showed various range of MIC/MBC on the test bacteria, had also similarly perform a different degree of inhibitory and fungicidal efficacy with MIC/ MFC ranges from 6.25% to 50% on C. albicans.
Looking at the lowest MIC value (6.25%), all the different extracts of Punica granatum showed a promising inhibitory effect on S. aureus (MTCC2001), while their bactericidal concentration was found to be 12.5% with methicillin and water extracts. Moreover, from all the test organisms used, Punica granatum relatively demonstrated better potential with MIC between 6.25% and 12% for all its extracts against on both standard and
drug resistant Streptococcus pneumonia.
All extracts of Punica granatum showed a strong inhibitory and bactericidal activity against S. pneumonia (MTCC5954) and drug resistant S.pneumonia with the MIC and MBC ranged from 6.25% to 12.5%, respectively. On the other hand, all the crude extracts of Punica granatum showed MIC ranged from the lowest concentration value 6.25% to 25%, but their MBC reaches to the highest dilution point (50%), against MRSA, S. boydii (MTCC7704) and C. albicans.
The minimum concentration that was enough to inhibit the growth of bacteria with MIC 6.25% was more effective against gram positive than gram negative with all different extractions of Punica granatum. There was no any inhibitory and bactericidal activity exhibited towards on both E. coli (MTCC8650) and E.coli (drug resistant) gram negative bacteria for water extracts of Punica granatum.
Evaluation of synergetic effect of plant extract and pure antibiotics
Evaluation of synergetic effect whether the plant crude extracts can exert synergism when tested in combination with the selected commercial antibiotics has been investigated in the study and it's presented below in the Figure 1.
MRSA was showed for synergetic effect for the combination of Punica granatum chloroform extract with both amoxicillin and vancomycin where their inhibition zone recorded 18 mm and 24 mm in diameter, respectively.
One of the crude extracts (Punica granatum acetone) was subjected for purification the bioactive components by column chromatography using silica gel (60-120 mesh). Apiece of cotton was placed at the bottom of the column. Elution was carried out using different solvents (petroleum ether, mixture of petroleum: acetone (ratio), acetone) as per method recommended by  with some modifications. Silica gel packed with solvents in the column.
The crude Punica granatum acetone extract was loaded at the top of the column and successive fractionated elutes were collected separately in different clean pre weighted test tubes. Eluents were evaporated at room temperature. The fractions were checked for their antibacterial activity against S. aureus and E. coli (drug resistance) by agar well diffusion method. The fractions with petroleum ether were active against S. aureus, fractions eluted by the mixture petroleum ether: acetone (1:6) and acetone showed antibacterial activity against both targeted bacteria. Therefore, this implicated the plant may contain different constituents of polar and non-polar compounds.
The antimicrobial effects of Punica granatum were previously studied. Indeed, it is reported that leaves of Punica granatum are widely used as antimicrobial agents . Moreover, in this study all the four different solvent extracts tested also showed antibacterial and antifungal activities. This may be due to the presence of some metabolic toxins or new bioactive compounds  and its antibacterial activity could indicate the presence of wide spectrum secondary metabolites than tannins. The study also showed that, all the extracts of the same plant possesses strong antibacterial activities against on both selected Grampositive and Gram-negative bacteria, and similarly it was also reported [24,25] that it had the ability to inhibit the activity of drug resistant S. aureus and E. coli. However, it was in contradictory to the study  that the water extracts of the Punica granatum didn't show any antibacterial activity against the standard and drug resistant E.coli strain in our investigation. Hofling, et al.  reported that methanol extracts of Punica granatum showed antifungal activity against C. albicans. These results are in accordance to results obtained in the present study for C. albicans wherein antifungal activity was observed for all the four different solvent extracts. Subsequently, a multidisciplinary approach shall be conducted to identify the novel active constitutes which are specifically responsible for the antifungal activity, then to contribute the best solution to the current productivity crisis facing the scientific community engaged in drug discovery and development.
Punica granatum chloroform extract were measured for combined effects against to all pathogens used in the sensitivity test and specifically showed synergistic effect against MRSA. Data from the literature as well as our results reveal the great potential of the plant for therapeutic treatment, in spite of the fact that they have not been completely investigated. Therefore, more studies need to be conducted to search for new compounds. Once extracted, and before being used in new therapeutic treatments, they should have their toxicity test in vivo.
In conclusion, the results certainly demonstrated that the therapeutic potentials of Punica granatum leaf extracts in the control of resistant bacteria and fungus; which are becoming a threat to human life, in all the techniques applied to check its efficiency. An in vitro investigation of each crude extracts has also exhibited an inhibitory effect towards in all targeted microorganisms.
Conflict of Interest
The authors declared that there is no conflict of interest.