RESULT of inhibition varied ranging from (7.7+0.6 mm) to

RESULT

Antibacterial
activity of Croton macrostachyus stem
bark extract

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The
antibacterial effects of C. macrostachyus stem bark extracts showed
effective antibacterial activity against all the tested bacterial strains. The
diameter of the zone of inhibition varied ranging from (7.7+0.6 mm) to (17+1.0
mm) 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, which
were inhibited with a zone diameter of 17+1 mm, were S. aureus (standard) (with methanol extracts)
and the lowest inhibition zone (7.7+0.6
mm) was found against E. coli (clinical)
which is extracted by methanol. The antibiotic Chloramphenicol (positive
controls) was frequently high. DMSO, which was
the negative control had no inhibitory activity.

 

Table 1.
Antibacterial activity of the chloroform, methanol and water extracts of Croton. macrostachyus stem bark against clinical
and standard strains of S. aureus and
E. 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 are
significantly different at (P< 0.05). Determination of Minimum inhibitory concentration and Minimum bactericidal concentration The MIC value of  C. macrostachyus stem bark extracts  against the tested bacteria ranged from 62.5 mg/ml (Methanol extract of  C. macrostachyus  both on clinical and standard strains of  E. coli) to 500 mg/ml (water extract on the same bacteria). Methanol extract of C. macrostachyus stem bark showed least MIC value 62.5 mg/ml against E. coli (both on clinical and standard strains) while water extract showed 500 mg/ml against E. coli (clinical). S. aureus (clinical and standard) and E. coli (standard) showed comparatively efficient MIC value 125 mg/ml in chloroform and methanolextracts (Table 2). The MBC values, which were determined by sub-culturing the samples having dilution values of greater or equal to MIC values, were described in Table 2. The MBC values of the extracts ranged from 125 mg/ml (Methanol extract against E. coli, both clinical and standard) to 500.00 mg/ml (water extract against the growth of E. coli (standard and clinical) and chloroform extract against E. coli and S. aureus (clinical).   Table 2: MIC and MBC (mg/ml) of the chloroform 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;     DISCUSSION The antibacterial analysis was performed using the agar well diffusion and broth dilution methods. Each of the extract tested in the present study displayed antibacterial activity on all the bacterial strains tested. However differences were observed between antibacterial activities of the extracts. These differences could be due to the differences in the chemical composition of these extracts. In the present investigation, chloroform, methanol and water extracts of C. macrostachyus stem bark were evaluated for examination of their antibacterial activity against Gram negative (E. coli) and Gram positive (S. aureus) bacteria, which was regarded as human pathogenic microorganisms. Antibacterial activity of each plant extract was tested by agar well diffusion and broth dilution (MIC) methods. The extracts from C. macrostachyus stem bark persuaded growth inhibition against all the studied bacterial pathogens. Our results illustrated that between the bacterial strains there was variation in susceptibility to extracts. This may be due to the antibacterial effect of the extract depends on the bacterial strain and the extraction solvent used to extract the phytochemicals which contain antibacterial effect from the medicinal plant.   In this 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 is reported that Gram positive bacteria should be more susceptible since they have only an outer peptidoglycan layer which is not an efficient barrier (Lulekal et al., 2014; Karou et al., 2005). Gram-negative bacteria have an outer phospholipidic membrane that make the cell wall impermeable to lipophilic solutes, while the porines contain a selective barrier to hydrophilic solutes with an exclusion limit of about 600 Da (Karou et al., 2005). The periplasmic space of Gram-negative bacteria also contains enzymes, which are able to break foreign molecules and appears to be less susceptible to plant extracts than the gram positive one. Numerous results confirmed this explanation, thus some plant extracts were found to be more active 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 to development of resistance in the clinical isolated.   Chloroform extract of the C. macrostachyus stem bark was the second strong extract for its antibacterial activity this is in agreement with Taye et al. (2011). But C. macrostachyus water extract had lower activity against the all bacteria tested. This indicates in comparison to water, the active ingredient which inhibits the growth of bacteria 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 reported to have strong antimicrobial activity against several gram positive and gram negative human pathogenic bacteria (Thatoi et al., 2008) and as stated by Dabur et al., 2007, the water extracts of A. nilotica, J. zeylanica, L. camera and S. asoca, were found to be the most active against different bacteria as well as fungal pathogens. It is clear that the effectiveness of the extracts largely depends on the type of solvent used. The organic extracts provided more powerful antimicrobial activity as compared to aqueous extracts. This observation clearly indicates that the existence of non-polar residues in the extracts which have higher both bactericidal and bacteristatic abilities. Thatoi et al., 2008, mentioned that most of the antibiotic compounds already identified in plants are reportedly aromatic or saturated organic molecules which can easily solubilized in organic solvents. Similar results showing that the alcoholic extract having the best antimicrobial activity is also reported by Antarasen and AmlaBatra (2012) in Melia azedarach leaf extracts   The antimicrobial 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 the MBC values suggesting that the extracts were bacteriostatic at lower concentration but bactericidal at higher (Maji et al., 2010; Antarasen and Amlabatra, 2012). Minimum inhibitory concentration values of 62.5–500 mg/ml. However Jackie et al. (2016) reported MIC value range from 125-500mg/m of C. macrostachyus ethanol extract against selected human pathogens.When testing methanol extracts of C. macrostachyus leaves and roots Wagate and colleagues found MICs from 15.6  to 250 mg/ml against three bacteria, E. coli, Bacillus cereus, and Pseudomonas aeruginosa.     CONCLUSION   From our investigation, it is concluded that the active antibacterial present in the stem bark of C. macrostachyus were methanol and chloroform soluble. The active ingredients contained in extract of chloroform are quite effective against standard strains of S. aureus and E. coli along with activity against the remaining whereas the activity in methanol extract showed efficacious results against all the tested organisms.Further studies should be conducted with different extraction solvents and toxicity and phytochemical analysis must be performed on these plants to use as sources and templates for the synthesis of drugs to control disease-causing bacteria. ACKNOWLEDGMENT  The authors of this paper are thankful to office of the vice president for research and community service, university of Gondar for their modest financial assistances.   Conflict of Interest The authors have not declared any conflict of interests.