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Research Paper
Abstract
Extract of the leaf of Olea europaea was investigated for antibacterial activity against six pathogenic bacteria. The leaf
extract was prepared using water and methanol in a cold extraction method. The test bacteria (Escherichia coli, Klebsiella
pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Salmonella enterica) were
isolated from a refused dump site in Akure, Ondo State, Nigeria. The extracts were found to be effective against all the test
isolates. The methanolic extract, at a concentration of 50 mg/mL, exhibited the highest inhibitory potential on Salmonella
enterica and Escherichia coli with a zone of inhibition of 13.0 mm and 10.5 mm respectively while the aqueous extract had
the highest inhibitory potential on Enterococcus faecalis and Escherichia coli with an inhibitory zone of 5.0 mm.
The phytochemical screening of the leaf extract revealed the presence of tannins, oleuropein, flavanoids, phlobatannins and
alkaloids in contrast to the absence of cardiac glycosides and saponins. The Minimum Inhibitory Concentration (MIC)
obtained with the methanolic leaf extracts of the plant was from 3.125 to 12.5 mg/mL and 12.5 to 25 mg/mL for the
aqueous extract. These results therefore inferred the antibacterial efficacy of the Olea europaea.
Keywords: Antibacterial Activity, Minimum Inhibitory Concentration, Olea europaea, Refused Dump Bacteria
1. Introduction
The World Health Organisation (WHO, 2008) reported
that for every one hour, over 1,500 people die from an
infectious disease of which more than half of them are
children under five years old. It was also reported by The
American College of Emergency Physicians that infectious
diseases are the third and second leading cause of death in
the U.S.A. and worldwide. It has been observed that 19%
of deaths in developed countries and 43% of deaths in
third world (developing) countries are due to infectious
diseases; this is related to the eruption of drug resistant
microorganisms and the emergence of unknown disease
causing microbes (Fausi, 2005).
Traditional medicine comprises of medical knowledge systems that developed over generations within various societies before the era of modern medicine. The WHO defines
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(Mallikhaarjuna et al, 2007). However, these defensive substances give plants their medicinal value which is employed in maintaining the health of individuals and communities (Akharaiyi & Boboye, 2010).
Recently microorganisms have developed resistance to
certain antibiotics. This has led to the combination of two
or more antibiotics (Synergism). However, the combination of these antibiotics is not giving an adequate solution to
the antimicrobial resistance. Based on the antimicrobial
resistance, several researches on the antimicrobial activities and photochemical evaluations of medicinal plants have
been done to know the exact bioactive components present
in a particular plant which have been found effective in the
treatment of diseases. These medicinal plants are promiseng in the treatment of diseases. The objectives of this work
are to assay for antibacterial activity of the extract of Olea
europaea, determine the phyto-chemical component of the
extract and determine the Minimum Inhibitory Concentration (MIC) of the leaf extract.
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C for six weeks after which they were milled into fine
powder with a grinding machine in the Department of Animal Production and Health, Federal University of Technology, Akure. A 500 g of the fine powder were soaked in
300 ml water and 300 ml methanol separately contained in
two different transparent plastic bowls. The bowls were
covered with their respective covers and allowed to stand
for 72 hr. Afterwards the solution was sieved with muslin
cloth. The filtrate was collected into a beaker and subjected to drying in a rotary evaporator, after which the leaf
extract was used for other analyses.
2.5. Phytochemical Screening of the Leaf Extract
Phytochemicals present in the leaf extract of Olea europaea prepared above were qualitatively determined. The
chemical components tested for were alkaloids, oleuropein
(irioids), saponins, flavanoids phlobatannins, tannins, anthraquinones and cardiac glycosides. They were determined
using standard chemical techniques.
2.6. Determination of Antibacterial Activity of the Leaf
Extract
The conventional agar diffusion method was used, with the
leaf extract at concentrations of 15, 25, 30 and 50 mg/ml.
Incubation of the test bacteria seeded into the nutrient agar
was carried out at 37 oC for 24 hours. The zones of
inhibition produced by the extract after incubation with the
test microbes were measured. Each concentration was
tested in triplicates.
2.7. Determination of Minimum Inhibitory Concentrations of the Leaf Extract
Four concentrations i.e. 3.12, 6.25, 12.5 and 25 mg/ml of
the leaf extract were prepared and introduced into different
wells bored into the medium (NA) containing the test microorganisms. The inoculated NA plates were incubated at
37 oC for 24 hr and zone of inhibition was measured in
millimetre. Minimum inhibitory concentration of the extract for each bacterium was determined by the lowest concentration of the leaf extract that showed least zone of inhibition.
2.8. Antibiotics Sensitivity Test
The disc diffusion method was used to determine the antibacterial activities of standard or commercially produced
antibiotics against the test microorganisms. A paper disc
impregnated with antibiotics was placed with the aid of a
sterile forceps on the surface of solidified agar (NA) plates
already inoculated with 18 hr old broth culture of each of
the microorganisms. The plates were incubated at 37 oC
for 24 hr after which zone of inhibition around each of the
antibiotics was viewed and measured.
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3. Results
3.1. Laboratory Identification of Test Microorganisms
The observed morphological and biochemical properties of
the isolated bacteria are presented in Table 1. All the microorganisms were non-motile with the exception of Escherichia coli and Salmonella enterica; the bacteria were positive to catalase reaction; and were able to ferment galactose, mannitol and glucose while only Staphylococcus aureus and Salmonella enterica were unable to ferment sucrose. The morphological tests revealed that all the microorganisms with the exception of Staphylococcus aureus and
Enterococcus faecalis are rod shaped and positive to Gram
reaction.
3.2. Phytochemical Quality of the Leaf Extracts
The results (Table 2) of the phytochemical screening of the
leaf extract shows that tannins, oleuropein, flavanoids, phlobatannins and traces of alkaloids were present while cardiac glycosides, saponins were absent in the water and methanol leaf extract of Olea europaea.
3.3. Antibacterial Activity of Olea europaea
The antibacterial activity of the water and the methanol
extract of Olea europaea leaf on the test organisms at different concentration is shown in Figure 1. The plant extract
had inhibitory effect on the entire test organisms. The methanolic extract exhibited the highest inhibitory potential on
Salmonella enterica and Escherichia coli with a zone of
inhibition of 13 mm and 10.5 mm respectively at a concentration of 50 mg/ml while the aqueous extract has the highest inhibitory potential on Enterococcus faecalis and Escherichia coli with a zone of inhibition of 5 mm at a concentration of 50 mg/mL. The DMSO that served as the control
did not show any activity against the test microorganisms.
3.4. Minimum Inhibitory Concentrations of the Leaf Ext-
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Table 1. Morphological and Biochemical Characteristics of the Bacteria Isolated From a Dumpsite in Akure, Nigeria
Bacteria
Characteristics
Gram Reaction
Shape
Motility
Catalase
Coagulase
Oxidase
Indole
Escherichia
coli
-
Klebsiella
pneumoniae
-
Salmonella
enterica
-
Enterococcus
faecalis
+
Pseudomonas
aeruginosa
-
Staph.
aureus
+
Short Rod
+
+
+
Rod
+
-
Rod
+
+
-
Cocci
+
-
Short Rod
+
+
-
Cocci
+
+
-
AG
AG
AG
AG
AG
AG
AG
AG
AG
A
A
AG
AG
-
AG
AG
A
AG
AG
AG
AG
AG
AG
-
AG
AG
AG
AG
AG
Fermentation of Sugar
Glucose
Sucrose
Galactose
Mannitol
Lactose
Key: - = Negative, + = Positive, AG = Acid and Gas Production, A= Acid Production only
Table 2. Phytochemical Quality of The Leaf Extract of Olea
europaea Extract
Phyto-chemical Group
Oleuropein (Irioids)
Tannins
Flavanoids
Phlobatannins
Alkaloids
Saponins
Methanol
Water
+
+
+
+
+
-
+
+
+
+
+
-
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a
Gram positive bacteria:
Z one of Inhibition (m m)
16
14
Staphylococcus aureus
12
Enterococcus faecalis
10
8
6
4
2
0
AUG
AMX
ERY
TET
CXC
GEN
COT
CHL
Antibiotic
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b
Gram negative bacteria:
18
Escherichia coli
Klebsiella pneumoniae
Salmonella enterica
Pseudomonas aeruginosa
16
Zone of Inhibition (mm)
14
12
10
8
6
4
2
0
AUG
AMX
TET
GEN
COT
AMX
OFL
NIT
Antibiotic
4. Discussion
The results obtained in this study revealed antimicrobial
efficacy of the aqueous and methanolic leaf extract of Olea
europaea on test isolates. The inhibitory activities exhibited by Olea europaea on isolates tested support its use as
Furthermore, the minimum inhibitory concentrations observed from the methanolic leaf extracts of the leaf are in the
range of 3.125-12.5 and 12.5-25 mg/ml for the aqueous extract. It can be deduced from the result recorded in Figures
1 and 2 that Salmonella enterica and E. coli have the
widest zones of inhibition and lowest minimum inhibitory
concentrations while Enterococcus faecalis at a concentration of 15 mg/ml of the methanolic extract has the lowest
zone of inhibition and the highest minimum inhibitory
concentration. This result correlates with the report that
microorganisms vary in their degree of susceptibility against antimicrobial agents and that antimicrobial agents with
low activity against an organism have high MIC while an
antimicrobial with high activity have a low MIC (Banso &
Ayodele, 2005).
Staphylococcus aureus is frequently connected to cases of
bacteraemia, septicaemia, endocarditis, osteomyelitis, furuncle, etc. It is also frequently involved in both nosocomial
and community acquired infections. The successful inhibition of this bacteria and its contemporary aetiology of
gastroenteritis (E. coli) is a good development, especially
when the records of its resistance to various conventional
antibiotics is considered (Voss, 1996; Wiedemann, 1996
and Ayliffe, 1997). This extract could therefore be of use
in management of opportunistic infections in HIV/AIDS
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5. Conclusion
This work has vividly shown that both methanolic and aqueous extracts of the Olea europaea have antibacterial
property against the six potential pathogens of public health importance tested in this research. Similarly, the extract
showed appreciable level of potency against the commonest aetiology of enteric fever which have been recorded to
cause 10-15% mortality rate in developing countries. Both
extracts were most effective against different bacteria at 50
mg/mL. The MIC values were however lower 50 mg/mL.
These concentrations could be put into fixed dosage combination therapy for treating the infections caused by bacteria associated with refused dump particularly Salmonella
enterica and Escherichia coli and Enterococcus faecalis.
Extracts from this Olea europaea are already in use by the
traditional medicine practitioners in Nigeria, though in either methanolic or aqueous form. By virtue of high activity
indices above unitary value even in crude forms, the extracts have more promising therapeutic advantages than the
likes of gentamicin, ciprofloxacin, amoxicillin, augmentin
and its relations when refined to produce antibiotics. The
observed antimicrobial effects of this medicinal plants on
the organisms tested, though in-vitro appear interesting
and promising and may be effective as a potential source
of novel antimicrobial drugs.
Acknowledgement
I do appreciate the Federal University of Technology
Akure (FUTA) Nigeria, for the financial support given to
me during the execution of this project. I acknowledge the
useful contributions given to the success of this investiga-
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tion particularly by Omiyefa, O. O. and various staff members of Microbiology and Biology Departments of FUTA,
particularly Professors F.C. Adetuyi and C.O. Adedire. I
am grateful for the assistance of Dr. F.O. Olukunle and
Prof. B. Boboye in the preparation of this manuscript for
publication.
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