All parts of Melia azedarach L. are found to possess numerous medicinal properties such as antioxidant,1,2 antibacterial.3 The antibacterial potential of M. Azedarach L. was tested using crude leave, flower and fruits seed extracts against pathogenic bacteria strains.3 Aim of this study was to evaluate antibacterial effects of Melia azedarach fruit extracts against some isolated pathogenic bacteria.
Materials and Methods
Melia azedrach fruits were collected from different sites of Baqubah, Diyala, Iraq. The fruit crushed and grind to a powder. Of powder 25 g of plant fruit seed were filled in the thimble and extracted successively with 300 mL of methanol using a Soxhlet extractor for 72 hours. The extract was concentrated using rotary evaporator, after complete solvent evaporation. The solvent extract was weighed and preserved at 4°C in airtight bottles until use. The powder of plant fruit seed were mixed thoroughly with 300 mL of distilled water in a beaker on magnetic stirrer for 24 h. Then filtered through Whatman No.1 filter paper. The extract was concentrated using rotary evaporator, after complete solvent evaporation. The solvent extract was weighed and preserved at 4°C in airtight bottles until use.
In the study of the antibacterial activity, both extracts were diluted in dimethyl sulfoxide (DMSO). The concentrations used in this experiment were 50, 100, and 200 mg/mL.
Antimicrobial activity was tested using a modified disc diffusion assay (DDA) method originally described by Bauer4 and Ncube and colleagues.5 The inoculums for each microorganism were prepared from broth culture (105 CFU/mL). A loop of culture from the nutrient agar (NA) slant stock was cultured in Luria-Bertani (LB) medium over night and spread with a sterile swab into Petri-plates. Sterile disc (6 mm) impregnated with the plant extracts (50, 100 and 200 mg/mL) were placed on the cultured plates and incubated for 24 h at 37°C. The solvent loaded disc without extracts in it served as control in the study. Clear inhibition zones around discs indicated the presence of antimicrobial activity. All data on antimicrobial activity were average of triplicate. Antimicrobial activities of both extracts were evaluated by the agar well diffusion method. For agar well diffusion method,6,7 antimicrobial susceptibility was tested on solid agar – agar media in petri plates. Nutrient agar (NA) plates were swabbed (Sterile cotton swabs) with 8 hours old – broth culture of respective bacteria. Well (10 mm diameter and about 2 cm a part) were made in each of these plates using sterile cork borer. Stock solution of fruit extracts was prepared at a concentration of 50 mg/mL, 100; 200 mg/mL in aqueous and methanolic extracts. About 100 μL of different concentration of plant solvents extracts were added sterile syringe into the wells and allowed to diffuse at room temperature for 2h. The plates were incubated at 37°C for 24 h. The diameter of the inhibition zone (mm) was measured triplicate were maintained and the experiment was repeated thrice, for each replicates the reading were taken in three different fixed directions and the average values were recorded.
The following bacterial strains were used as test organisms: Staphylococcus aureus, Bacillus subtilis, E. coli, Pseudomonas aeruginosa, Klibsiella and Proteus. All the bacterial strains were obtained from Department of Microbiology, College of Veterinary Medicine, University of Diyala, Iraq.
All values are expressed as the mean ± the standard error of the mean (SEM). The data were analyzed by using one way analysis of variance ANOVA, and then the test of the least significant differences between the means of inhibitory zones.8 The significant level of test was P<0.05.
The results revealed that both alcohol and aqueous extracts are potent antimicrobial against all the microorganisms studied.
The results revealed that aqueous extract of Melia azedarach fruit in well and disc methods showed no significance in inhibitory zone against E. coli. But there was higher significant difference at 50 mg/mL concentration in disc method comparing with well method. Against Pseudomonas aeruginosa in well method there was significant difference in inhibition at 200 mg/mL in comparison with 50 mg/mL. While in disc method the inhibitory zone was significance at 100 and 200 mg/mL comparing with 50 mg/mL. When compared well and disc methods there was significant difference at 100, and 200 mg/mL. In case of Klebsiella in well method there was significant difference in inhibition at 200 mg/mL in comparison with 50 mg/mL. While in disc method there was significant difference in inhibition at 100 and 200 mg/mL in comparison with 50 mg/mL. In comparison between well and disc method Klebsiella did not show any significant difference. While in case of Proteus in well method there were significant difference at 100, and 200 mg/mL in comparison with 50 mg/mL. In disc method there was significant difference at 200 mg/mL in comparison with 50 mg/mL. In comparison between well with disc method Proteus showed no significant difference. Bacillus in well and disc methods did not show any significant difference. In comparison between disc sand well methods also there was no significant difference. Against staphylococcus aureus in well method there was significant difference at 100 and 200 mg/mL in comparison with 50 mg/mL. In disc method the significance was at 100 and 200 mg/mL in comparison with 50 mg/mL. In comparison between well and disc method there was no significant difference (Table 1).
The results revealed that in alcohol extract of Melia azedarach fruits showed no significant inhibitory difference against E. Coli; Bacillus subtilis and Klebsiella in both well and disc methods. In disc method Pseudomonas aeruginosa showed a significant inhibitory difference at 200 mg/mL in comparison with 50, and 100 mg/mL. In comparison between well and disc there was no significant difference in sensitivity of Pseudomonas aeruginosa against alcohol extract of Melia azedarach fruit. Against Proteus in well and disc methods, there was significant inhibitory difference at 200 mg/mL in comparison with 50 mg/mL. In comparison between well with disc method there was no significant difference against staph aureus there was significant inhibitory difference at 200 mg/mL in comparison with 50 mg/mL in disc method (Table 2).
In comparison between alcohol and aqueous extract, the results revealed that in case E. coli; Proteus; Bacillus and Staphylococcus aureus none. In case of Klibsiella in well method none. In disc method significant at 50 mg/mL. In case of Pseudomonas aeruginosa in disc method at 50 mg/mL (Tables 3 and 4).
Among the different solvents extracts studied methanol and ethanol showed high degree of inhibition followed by petroleum ether and aqueous extract.9 Methanol extract showed maximum inhibition zone in E. coli and B. cereus, more specifically, aqueous extract represented higher susceptibility to all bacterial strains.9
Dichloromethane leaf extract of M. azedarach was found to be more effective against gram positive than gram negative bacterial strains, while ethanol extract inhibited the growth of three gram positive and two gram negative strains, ethyl acetate, methanolic fraction and aqueous extract were found to be effective against all tested bacterial strains.10
Results of this study confirm that the fruit extracts showed an antibacterial effect. E. coli and Bacillus were the least sensitive bacteria Pseudomonas, Staphylococcus, Proteus and Klibsiella were the sensitive bacterial strains to M. azedarach fruit aqueous and alcohol extract. No differences were observed between two methods well and disc; and between extract aqueous and alcohol.
Results of this study strongly confirm that the fruit extracts could be effective antibacterial compounds.