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Dada / Journal of Biology (2013), Vol. 01, Issue 06, pp.

118-124

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Research Paper

Antibacterial Activities of Olea europaea Leaf Extract on


Some Bacteria Isolated from a Refused Dump Site in
Akure, Nigeria
E.O. Dada
Department of Microbiology, P.M.B. 704, Federal University of Technology, Akure, Ondo State, Nigeria
E-Mail: dadaoluyemi5@gmail.com.

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

traditional medicine as the health practices, approaches,


knowledge and beliefs incorporating plant, animal and mineral-based medicines, spiritual therapies, manual techniques and exercises, applied singularly or in combination to
diagnose, treat and prevent illnesses or maintain well-being. Traditional medicine includes disciplines such as herbals, ethnomedicine, ethnobotany and medical anthropology. The WHO reported that in many developed countries,
70% to 80% of the population has used some form of
alternative or complementary medicine e.g. acupuncture,
which is an alternative medicine methodology that originated in ancient China that treats patients by manipulating
thin, solid needles that have been inserted into acupuncture
points in the skin. In some Asian and African countries,
80% of the population depend on traditional medicine for
primary health care (WHO, 2008). Traditional practices in-

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clude herbal, ayurvedic medicine which is a Hindu system


of traditional medicine, native to India; Unani, a form of
traditional medicine widely practiced in South Asia, it is
based on the teachings of Greek physician Hippocrates and
Roman physician Galen (Leventin & McMahon, 2003).
Traditional medicine like orthodox medicine has its own
methods and techniques of application which however
aims at healing diseases (Wurochekke et al, 2008).
The treatment and control of disease by the use of available medicinal plant as a loyalty will continue to play a significant role in medical healthcare implementation in the
developing countries of the world. Nearly all cultures and
civilization from ancient times to the present day have
fully or partially depended on herbal medicine because of
their effectiveness, affordability, availability, low toxicity
and acceptability. Due to the ineffectiveness of most orthodox drugs associated with microbial resistance to available
therapeutic agents, most patients are seen in medical centres than ever. The problems of antimicrobial resistance has
led to the resurgence of interest in herbal products as sources of novel compounds to suppress or possibly eradicate
the ever increasing problems of emergence of newer diseases thought to be brought under control (Wurochekke et al,
2008).
Medicinal plants are backbone of traditional medicine and
the findings of the healing power in plants is an ancient
idea. As a matter of fact, many of the herbs and spices used by humans to season food, yield useful medicinal compounds and many familiar medications of the twentieth
century were developed from ancient healing traditions
that treated health problems with specific plants. Today,
science has identified the medicinal properties of a large
number of botanicals and their healing components have
been extracted and analysed. Many plant components are
now synthesised in large laboratories for use in pharmaceutical preparations. For example, vincristine (a vinca alkaloid), that is obtained from the leaf Catharanthus roseus
(Madagascar periwinkle), is a mitotic inhibitor used in
cancer chemotherapy. Also digitalis: a drug prepared from
the plant, Digitalis purpurea (foxglove leaves), contains
substances that stimulate the heart muscle, while ephedrine
(an alkaloid drug derived from the plant, Ephedra fragilis)
used to increase the activity of noradrenalin on adrenergic
receptors, were all originally discovered through research
plants (Odugbemi, 2006).
Plants have limitless ability to synthesize aromatic substances, mainly as secondary metabolite, of which at least
12,000 substances have been isolated; a number that is
10% of the total. The synthesized aromatic substance is
used by plants as defensive molecule against predations by
microorganisms, insects and herbivores. Some of these
molecules are responsible for plants odours (terpenoids),
pigmentation (tannin and quinines) and flavour (capsaicin)

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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.

2. Materials and Methods


2.1. Samples
Fresh leaves of the Olea europaea used in this work were
collected from the side of Akure-Ilesa Road in Akure,
Nigeria, where they were found growing naturally (Plate
1). The soil sample used for this project was obtained from
the refused dump site in Sijuade, Akure, Nigeria. Test bacteria were isolated from the soil sample.
2.2. Growth Medium
The medium used was Nutrient Agar (NA). It was prepared by dissolving 2.8 g of the NA powder in 100 ml of distilled water. All the reagents used for identification tests
were obtained from the laboratory of Microbiology Department in the Federal University Technology Akure, Nigeria.
2.3. Isolation and Identification of Test Bacteria
The bacteria were isolated from the soil sample using conventional microbiological techniques and biochemical tests
according to Olutiola et al (2000). The tests carried out on
the isolated bacteria were Gram staining, motility, sugar
fermentation, catalase, oxidase, coagulase and indole tests.
The sugars used for the fermentation test were glucose,
sucrose, galactose, mannitol and lactose. After Gram staining, the Gram reaction and shapes of the cells were examined under a light microscope with at oil immersion objective lens at a magnification of 100X.
2.4. Preparation of Leaf Extract
The fresh leaves of Olea europaea were air dried at 25-28

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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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2.9. Standardization of Bacterial Samples


Ten millilitres of 18 hr old broth culture of each of the test
microbes was centrifuged at 2000 rpm for 10 min. It was
then decanted to retain residual cells and 10 mL of normal
saline was added to it. It was then centrifuged again to
wash the wells. This was done three more times. The washed cells were stored in 15 mL normal saline. Tubes of sterile distilled water were labelled A-C and serial dilution
was made from the washed organisms using sterile water
and separate sterile pipettes. From the tubes A-C, 1 mL
was drawn from each dilution into sterile Petri dish and
nutrient agar was poured into it allowed to solidify. The
plates were then incubated at 37 oC for 24 hr. This was
carried out to determine the colony forming unit per mL.

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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Dada / Journal of Biology (2013), Vol. 01, Issue 06, pp. 118-124

ract of Olea europaea


The MIC of the aqueous and methanolic leaf extracts of
the bacterial isolates is presented in Figure 2. The MIC observed from the methanolic and aqueous leaf extract is
3.125-12.5 and 12.5-25 mg/mL respectively which thereofre shows that the plant extracts were effective on the test
bacteria.

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ve and resistant to certain conventional antibiotics with the


zones of inhibition ranging from 4.0 mm to 14.0 mm. They
were both sensitive to amoxicillin, erythromycin, tetracycline, gentamicin, cotrimoxazole and chloramphenicol. They were both resistant to augmentin and cloxacillin. Also
for the Gram negative bacterial isolates, all the microorganisms tested were sensitive to tetracycline and resistant to
cotrimoxazole. Also for the Gram negative bacterial isola-

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

+
+
+
+
+
-

+
+
+
+
+
-

Key: + : Detectable; : Not detectable

3.5. Antibiotics Sensitivity Profile of the Test Bacteria

Figure 1. Antibacterial Activity of the Leaf Extract of Olea europaea


at Various Concentrations

Results of industrially prepared antibiotics disc of different


concentrations tested against the bacterial isolates are
shown in Figure 3 (a, b) for both Gram positive and Gram
negative bacteria. Staphylococcus aureus and Enterococcus faecalis which are Gram positive bacteria; were sensiti-

tes, all the microbes were sensitive to tetracycline and


resistant to cotrimoxazole. Nalidixic acid and gentamicin
were found to be effective on 75% of the Gram negative
bacteria while ofloxacin and amoxicillin were found to be
effective on 50% of the test isolates.

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Dada / Journal of Biology (2013), Vol. 01, Issue 06, pp. 118-124

Figure 2. Minimum Inhibitory Concentrations of the Methanolic


and Water Extracts of Olea europaea

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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an antibacterial agent. This is in agreement with previous


studies that it is an anti-inflammatory and antibacterial agent (Chukwujekwu et al, 2005; Ilias et al, 2011 and Mahjoub et al, 2011). In this study, the methanolic leaf extracts
of Olea europaea inhibited all the bacteria examined while
the aqueous leaf extracts of Olea europaea were noninhibitory to E. coli, Salmonella enterica and Enterococcus
faecalis at a concentration of 15 mg/mL. This data means
that methanol has extracted the active ingredient/s more
than the water from the plant used in this project. It is
equally similar to the findings of Obi & Onuoha (2000),
who documented that alcohol as the best solvent for the
extraction of active ingredients of plant of medical importance. The effect of diffusion rate of the antibiotics have
been shown to affect antimicrobial property of antibiotics;
in the same vein the phytochemicals in the Olea europaea,
appeared to have moved freely within the agar matrix and
hence the wider zones of inhibition exhibited by the methanolic and water extracts of Olea europaea (Cowan,
1999).
In addition this work reveals that the leaves of the plant,
Olea europaea, contained bioactive agents which are connected with antimicrobial properties in plants. These agents are oleuropein (a phenolic compound), phlobatannins,
flavonoids, tannins and traces of alkaloids. Research work
revealed that tannins from the barks, roots and leaves of
many plants are used to treat cells that have gone neoplastic (Duke & Wain, 1981).

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

Figure 3. Antibiotics Sensitivity Patterns of Bacteria Isolated from a


Dump Site in Akure, Nigeria
Legend: AUG: Augmetin, AMX: Amoxicillin, ERY: Erythromycin, TET:
Tetracycline, CXC: Cloxacillin, GEN: Gentamycin, COT: Cotrimoxazole,
CHL: Chloramphenicol, NAL: Nalidixic acid, OFL: Ofloxacin, NIT:
Nitrofurazone

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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involving these two isolates as documented by Sylvia et al


(2003).
Both methanolic and aqueous extracts could be put into
fixed dosage combination therapy for treating the Salmonella infection. This extracts is 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 aqueous and methanolic extracts have a high antibacterial effect
on the six potential pathogens of public health importance.
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 (Brooks et al, 2004). 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.

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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