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1.2 Determination of Antibacterial Activity
(a) Well-diffusion Assay
Azadirachta indica ethyl acetate extract gave maximum inhibition zone (10mm) against S. aureus, B. subtilis and P. aeruginosa while inactive against rest of strains (Fig.1) (plate no.1). The previous reported antimicrobial potential of Azadirachta indica leaves showed zone of inhibition of 20mm against E. faecalis and 7.1mm against C. albicans (Bohora et al., 2010). Zone of inhibition ranging from 0.8cm to 2cm was given by results of aqueous and alcoholic aleaf extract of Azadirachta indica against E. coli, S. aureus, S. Bacillus bacteria and Rhizopus fungi through cup-plate agar diffusion method (Gupta et al., 2013). The reason of difference between result of antibacterial activity of P. aeruginosa and E. coli was probably due to difference in method of solvent extraction (using soxhlet) and also due to variation in solvent used.
The Azadirachta indica chloroform extract was only active against S. aureus, giving 10mm inhibition zone around well while rest of all tested strains was seemed to be inactive (Fig.2) (plate no.2). The previously reported results were also same which was given by earlier workers for chloroform extract of Azadirachta indica against Pseudomonas sp., but the results deviated against E. coli and Klebsiella sp. (Shinde and Mulay, 2015; Zwetlana et al., 2014). The difference in results possibly due to the sources of microorganisms used.
The Azadirachta indica acetone extract gave 16mm maximum zone of inhibition against B. subtilis following 12mm against S. aureus, 11mm against E. coli, 10mm against P. aeruginosa, while the same extract was inactive against K. pneumoniae (Fig.3) (plate no.3). The similar result was also reported against Staphylococcus aureus Pseudomonas aeruginosa and Escherichia coli using acetone extract of Azadirachta indica (Shinde and Mulay, 2015; Edet, 2016).
The Mentha piperita E. acetate extract was inactive against all tested strain (Fig.1) (plate no.1) but the previously stated result of Mentha piperita L. indicated antibacterial activity against Bacillus sbubtilis, Streptococcus pneumonia, Staphylococcus aureus, Escherichia coli, Proteus vulgaris and Klebsiella pneumonia (Sujana et al., 2013). The variation from present results can be due to different extraction and experimental protocols.
The Mentha piperita chloroform extract was inactive against all tested strains of bacteria (Fig. 2) (plate no.2). Earlier reported study indicated that chloroform extract of Mentha piperita was active against Bacillus subtilis, Streptococcus pneumonia, Staphylococcus aureus, Escherichia coli, Proteus vulgaris, Klebsiella pneumonia (Sujana et al., 2013). The efficiency of plant extract against a particular pathogen is affected by various intrinsic and extrinsic factors may be the reason for the difference.
The acetone extract of Mentha piperita. gave 10mm inhibition zone against S. aureus but the respective was inactive against E. coli, K. Pneumonia, P. aeruginosa, B, subtilis (Fig.3) (plate no.3). The acetone extract of Mentha arvensis indicated the antimicrobial potential against B. cereus, Serratia sp., A. flavus and P. citrinum (Dhiman et al., 2016). The varying results between previous and present reported results may be due to the difference in tested species of plant and microorganisms employed in both study.
(b) Disc diffusion assay
Ethyl acetate extract of Azadirachta indica was found to be active against S. aureus (Fig. 4) (plate no.4). In previously reported result, respective test extracts of Azadirachta indica possesses significant antimicrobial activity against test microorganisms of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Proteus vulgaris and Klebsiella pneumonia (Latha et al., 2015). The reason of difference in results against E. coli and P. aeruginosa maybe the of difference in method used for preparation of plant extracts.
The chloroform extract of Azadirachta indica with comparison to pure solvent not show any obvious activity against all tested bacterial strains (Fig. 5) (plate no.5). Same results were also reported in previous study against P. aeruginosa and S. aureus (Al-Jadidi and Hossain, 2015).
Among acetone extracts, Azadirachta indicia gave 11mm and 10mm inhibition zone against B. subtilis and E. coli (Fig. 6) (plate no.6). The same results for such sample showed no antimicrobial activity against S. aureus and P. aeruginosa (Irshad et al., 2011). The understudy results also relate with earlier reported results against B. subtilis and E. coli. but as the results differed against S. aureus which is results of difference in experimental conditions.
Ethyl acetate extracted sample of Mentha piperita shown clear hallow against E. coli and K. pneumoniae but it was not active against P. aeruginosa, S. aureus and B. subtitles (Fig. 4) (plate no.4). Such result was also reported against E. coli and S. aureus but at 0.675 mg/ml concentration (Satmi and Hossain, 2016).
The chloroform sample from Mentha piperita was active against E. coli and K. pneumoniae while inactive against S. aureus, B. subtilis and against P. aeruginosa (Fig. 5) (plate no.5). In earlier reported results the activity was found against E. coli and S. aureus. The result was same against E. coli but the results deviated against S. aureus (Satmi and Hossain, 2016). Possibly the difference in result against S. aureus was due to difference in method of extraction of samples in previous (Soxhlet extractor) and present study.
The most obvious zone (14mm) was given by Mentha piperita. acetone extract against S. aureus (Fig. 6) (plate no.6). Same result was reported against Klebsiella pneumonia, Pseudomonas aeruginosa and Escherichia coli (Keskin and Toroglu, 2011).
1.3 Cytotoxicity potential of plants extracts against Brine shrimp larvae
Cytotoxicity potential of both plants were checked against brine shrimp larvae. Azadirachta indica plants showed maximum toxicity level (23.37%) with chloroform solvent while the Mentha piperita acetone extract showed least toxicity (9.15%) (Fig. 7). In reported results of water and methanol extracted samples of Azadirachta indica (Meliaceae) and Myrica salicifolia (Meliaceae) for brine shrimp the toxicity level was 101.26 ± 3.7 61.43 ± 2.9 (?g/ml) and 328.22 ± 10.9 320.17 ± 1.6 (?g/ml) respectively (Kirira et al., 2006). The toxicity study of under study Mentha piperita showed noticeable results among all extracts being tested.
CONCLUSION
Mostly biologically active compounds were found in E. acetate and acetone extracts of Azadirachta indica and acetone extract of Mentha. Using agar well diffusion assay chloroform and acetone samples of Azadirachta indica were found to be most active giving 16mm inhibition zone against B. subtilis. The disc diffusion assay was also used to determine the antimicrobial potential of respective plants in which ethyl acetate extracted sample Azadirachta indica indicated 12mm maximum zone of inhibition against B. subtilis. While acetone extracted sample of Mentha piperita exhibited 14mm maximum zone of inhibition. Brine shrimps microwell cytotoxicity assay was performed to check the cytotoxicity level of all the extracts. Mentha piperita acetone extract showed least toxicity (9.15%) while the Azadirachta indica plants showed maximum toxicity level (23.37%) with chloroform solvent.
RECOMMENDATION
The following recommendations could be made from the outcomes of this study. As only in vitro method was used in assessing the antimicrobial activity of the plant crude extracts, further investigations using bioassay guided fractionations are recommended to isolate and identify the pure compounds responsible for antibacterial activity of Azadirachta indica and Mentha piperita.
In addition, studies should be done in order to identify the active phytochemical constituents qualitatively in the respective plants extracts and also evaluate their efficiency in-vivo so that they can be made commercially. Furthermore, there is need for in-vivo trials to categorise those plants that are active and suitable for general use.