DISCUSSION layer which is an ineffective barrier

DISCUSSIONTheantibacterial analysis was performed using the agar well diffusion and broth dilution techniques.

Each of theextracts tested in the present study displayed antibacterial activity on allthe bacterial strains tested. However, differences were observed between antibacterialactivities of the extracts. These differences could be due to the variations inthe chemical composition of these extracts. Inthe present investigation, chloroform, methanol, and water extracts of C. macrostachyusstem bark were evaluated for examination of their antibacterial activityagainst Gram-negative (E.

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coli) andGram-positive (S. aureus) bacteria,which was regarded as important human pathogenic microorganisms. Antibacterialactivity of each plant extract was tested by agar well diffusion and broth dilution (MIC) methods. Theextracts from C. macrostachyus stembark persuaded growth inhibition against all the studied bacterial pathogens.Our results illustrated that between the bacterial strains there was variationin susceptibility to extracts. This may be due to the antibacterial effect ofthe extract depends on the bacterial strain and the extraction solvent used toextract the phytochemicals which contain antibacterial effect from themedicinal plant.

 Inthis study, C. macrostachyus stembark extracted by methanol has shown the highest inhibition zone (17+1)against S. aureus (standard)and the lowest inhibition zone was seen in E. coli (clinical). It isreported that Gram-positive bacteria should be more susceptible since they haveonly an outer peptidoglycan layer which is an ineffective barrier (Lulekal et al.

, 2014; Karou et al., 2005). Gram-negative bacteria havean outer phospholipidic membrane that makes the cell wall impermeable tolipophilic solutes, whereas the porines contain a selective barrier tohydrophilic solutes with an elimination limit of about 600 Da (Karou etal.

, 2005). Theperiplasmic space of Gram-negative bacteria also contains enzymes, which are ableto break strange molecules and become to be less susceptible to plant extractsthan the gram positive one. Numerous resultsconfirmed this explanation, thus some plant extracts were found to be moreactive against Gram-positive bacteria than against Gram-negatives (Kelmanson et al., 2000; Masika and Afolayane, 2002).The lowest inhibition zone was recorded against E. coli which is the clinical isolate; this may be due todevelopment of resistance in the clinical isolated.  Chloroformextract of the C. macrostachyus stembark was the second strong extract for its antibacterial activity and this isin agreement with Taye et al.

(2011).But C. macrostachyus water extracthad lower activity against the all bacteria tested. This indicates, incomparison to water, the active ingredient which inhibits the growth ofbacteria may dissolve better in methanol. However, Sendeku et al. (2015) reported chloroform extract from C. macrostachyus leaves shows significant antimicrobial activity.

Furthermore, water extract from leaves of P. acerifolium had been reportedto have strong antimicrobial activity against several gram positive and gramnegative human pathogenic bacteria (Thatoi etal., 2008) and as stated by Dabur et al., 2007, the water extracts of A.nilotica, J. zeylanica, L. camera and S.

asoca, were found tobe the most active against different bacteria as well as fungal pathogens. Itis clear that the effectiveness of the extracts largely depends on the type ofsolvent used to extract the phenolic compound from plants. The organic extractsprovided more powerful antimicrobial activity as compared to the waterextracts. This observation clearly indicates that the existence of non-polarresidues in the extracts which have higher both bactericidal and bacteriostaticabilities. Thatoi et al., 2008,mentioned that most of the antibiotic compounds already identified in plantsare reportedly aromatic or saturated organic molecules which can easilysolubilized in organic solvents.

Similar results showing that the alcoholicextract having the best antimicrobial activity is also reported by Antarasen and AmlaBatra (2012) inMelia azedarach leaf extracts Theantimicrobial analysis using the MIC value is been used by many researchers.In the present study the MIC value of the active C. macrostachyus stem bark extracts obtained were lower than theMBC values suggesting that the extracts were bacteriostatic at lower concentrationbut bactericidal at higher (Maji et al., 2010; Antarasen and Amlabatra,2012). Minimum inhibitory concentration values of 62.5–500 mg/ml. However Jackieet al. (2016) reported MIC value rangefrom 125-500mg/m of C.

macrostachyusethanol extract against selected human pathogens.When testing methanol extractsof C. macrostachyus leaves and rootsWagate and colleagues found MICs from 15.6 to 250 mg/ml against three bacteria, E.coli, Bacillus cereus, and Pseudomonas aeruginosa.

  CONCLUSION Fromour investigation, it is concluded that the active antibacterial present in thestem bark of C. macrostachyus were methanoland chloroform-soluble. The active ingredients contained in extract ofchloroform are quite effective against standard strains of S. aureus and E.

colialong with activity against the remaining whereas the activity in methanol extractshowed efficacious results against all the tested organisms.Further studiesshould be conducted with different extraction solvents and toxicity andphytochemical analysis must be performed on these plants to use as sources andtemplates for the synthesis of drugs to control disease-causing bacteria.ACKNOWLEDGMENT  Theauthors of this paper are thankful to the office of the vice president for researchand community service, University of Gondar for their modest financialassistance. Conflictof InterestTheauthors have not declared any conflict of interests.

      

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