RESULTAntibacterialactivity of Croton macrostachyus stembark extractTheantibacterial effects of C.
macrostachyus stem bark extracts showedeffective antibacterial activity against all the tested bacterial strains. Thediameter of the zone of inhibition varied ranging from (7.7+0.
6 mm) to (17+1.0mm) diameter (Table 1). All the water, chloroform and methanolic extracts of C.macrostachyus stem bark caused 7.7+0.
6 mm to 17+1 mm inhibition zones of bacterial growth. The bacteria, whichwere inhibited with a zone diameter of 17+1 mm, were S. aureus (standard) (with methanol extracts)and the lowest inhibition zone (7.7+0.
6mm) was found against E. coli (clinical)which is extracted by methanol. The antibiotic Chloramphenicol (positivecontrols) was frequently high. DMSO, which wasthe negative control had no inhibitory activity.
Table 1.Antibacterial activity of the chloroform, methanol and water extracts of Croton. macrostachyus stem bark against clinicaland standard strains of S. aureus andE. coli. Antibacterial Extraction Solvent Diameter of inhibition (mm) E. coli S. aureus clinical standard clinical standard C.
macrostachyus Chloroform 12+1.0a 13.0 +1.0ab 11.0+1.1a 16.0+3.
6b Methanol 12+1.0a 15.0+1.5b 12.
7+1.0a 17+1.0b Water 7.7+ 0.6a 10+1.
0a 8.67+0.6a 10+1.0a Chloramphenicol 39+1.0 42+1.0 42+1.0 39+1.
0 Values are means of triplicate determinations;Values within the same column followed by different superscripts aresignificantly different at (P< 0.05).Determinationof Minimum inhibitory concentration and Minimum bactericidal concentrationTheMIC value of C. macrostachyus stem bark extracts against the tested bacteria ranged from 62.5 mg/ml (Methanol extractof C.
macrostachyus both on clinical andstandard strains of E. coli) to 500 mg/ml (water extract on the same bacteria). Methanolextract of C. macrostachyus stem barkshowed least MIC value 62.5 mg/ml against E. coli (both on clinical and standard strains) whilewater extract showed 500 mg/ml against E.
coli (clinical). S. aureus (clinical and standard) and E.coli (standard) showedcomparatively efficient MIC value 125 mg/ml in chloroform and methanolextracts (Table2).TheMBC values, which were determined by sub-culturing the samples having dilutionvalues of greater or equal to MIC values, were described in Table 2. The MBCvalues of the extracts ranged from 125 mg/ml (Methanol extract against E. coli, both clinical and standard) to500.
00 mg/ml (water extract against the growth of E. coli (standard and clinical) and chloroform extract against E. coli and S. aureus (clinical). Table2: MIC and MBC (mg/ml) of thechloroform methanol and water extracts of C.macrostachyus stem bark extracts against clinical and standard strains of S. aureus and E. coli.
Bacteria Methanol Chloroform Water MIC MBC MIC MBC MIC MBC E. coli (clinical) 62.5 125 250 500 500 500 E. coli (standard) 62.
5 125 125 250 250 500 S. aureus(clinical) 125 250 250 500 125 250 S. aureus(standard) 125 250 125 250 250 250 MIC= minimum inhibitory concentration; MBC = minimum bactericidal concentration; DISCUSSIONTheantibacterial analysis was performed using the agar well diffusion and broth dilution methods. Each of theextract tested in the present study displayed antibacterial activity on all thebacterial strains tested.
However differences were observed betweenantibacterial activities of the extracts. These differences could be due to thedifferences in the 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. coli) andGram positive (S. aureus) bacteria,which was regarded as human pathogenic microorganisms. Antibacterial activityof 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, methanolic extract 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 not an efficient barrier (Lulekal et al., 2014; Karou et al.
, 2005). Gram-negative bacteria havean outer phospholipidic membrane that make the cell wall impermeable tolipophilic solutes, while the porines contain a selective barrier tohydrophilic solutes with an exclusion limit of about 600 Da (Karou etal., 2005).
Theperiplasmic space of Gram-negative bacteria also contains enzymes, which are ableto break foreign molecules and appears 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 this is inagreement with Taye et al. (2011).
ButC. macrostachyus water extract hadlower activity against the all bacteria tested. This indicates in comparison towater, the active ingredient which inhibits the growth of bacteria may dissolvebetter in methanol. However, Sendeku etal. (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. The organic extracts provided more powerful antimicrobialactivity as compared to aqueous extracts.
This observation clearly indicatesthat the existence of non-polar residues in the extracts which have higher bothbactericidal and bacteristatic abilities. Thatoi et al., 2008, mentioned that most of the antibiotic compoundsalready identified in plants are reportedly aromatic or saturated organicmolecules which can easily solubilized in organic solvents.
Similar resultsshowing that the alcoholic extract having the best antimicrobial activity isalso reported by Antarasen andAmlaBatra (2012) in Melia 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 office of the vice president for researchand community service, university of Gondar for their modest financialassistances.
Conflictof InterestTheauthors have not declared any conflict of interests.