AASCIT Journal of Bioscience

2017; 3(4): 29-34

http://www.aascit.org/journal/bioscience
ISSN: 2381-1250 (Print); ISSN: 2381-1269 (Online)

Antibacterial Activity of Persia

americana Leaf Extracts Against
Multidrug Resistant Bacterial
Isolates
Evbuomwan Lucky1, *, Inetianbor Jonathan2
1
Department of Microbiology, Faculty of Life Sciences, University of Benin, Benin City, Nigeria
2
Microbiology Department, Faculty of Pure and Applied Sciences, Federal University Wukari,
Wukari, Nigeria

Email address
evbuomwanlucky1@gmail.com (E. Lucky)
*
Keywords Corresponding author
Antibiotic,
Resistance,
Citation
Evbuomwan Lucky, Inetianbor Jonathan. Antibacterial Activity of Persia americana Leaf Extracts
Test Organisms,
Against Multidrug Resistant Bacterial Isolates. AASCIT Journal of Bioscience.
Zone of Inhibition,
Vol. 3, No. 4, 2017, pp. 29-34.
Phytochemical and Analysis
Abstract
The phytochemical and antibacterial properties of ethanol and aqueous extracts of
Persea americana against selected test organisms were carried out. Phytochemical
Received: June 28, 2017 screening was done using chemical methods while agar well diffusion method was used
Accepted: July 24, 2017 for antibacterial assay. Test organisms which were obtained from the University of
Published: August 30, 2017 Benin Teaching Hospital were Escherichia coli, Staphylococcus aureus, Pseudomonas
aeruginosa, Streptococcus pneumoniae and Klebsiella pneumonia. Phytochemical
screening revealed the presence of flavonoids, saponins, cardiac glycosides, quinones,
alkaloids, reducing sugar, proteins, steroids and terpenoids in both ethanolic and aqueous
extracts while tannin was however absent in both extracts. Varying degree of inhibition
zones was observed against the test organisms in the extracts. Inhibition zones ranged
from 15.0±0.0mm at 25mg/ml to 26±1.0mm at 200mg/ml against Escherichia coli in the
ethanol extract and in the aqueous extract, it ranged from 8.5±3.5mm at 100mg/ml to
11.0±0.0mm at 200mg/ml. There were no antibacterial activities at lower concentrations
of 12.5 mg/ml, 6.25 mg/ml, and 3.125 mg/ml. The minimum inhibitory concentrations
(MIC) against Escherichia coli in the ethanol extract was at 25mg/ml while in the
aqueous extracts, it was 100mg/ml against E. coli. The minimum bactericidal activity
against E. coli were 100mg/ml and 200mg/ml in the ethanol and aqueous extracts
respectively. The most resistant bacterium to conventional antibiotics was S. pneumoniae
(80%) while the most sensitive was K. pneumoniae (20% resistance). Susceptibility of
multiple drug resistant bacteria to Persea americana extract could be an alternative
source to conventional antibiotics for therapeutic uses.

1. Introduction
The use of plants and herb extract in the treatment of human ailments is a very ancient
art, a practice that has been passed on for generations and Scientists in Africa and other
developing countries are conducting research into local plants abundant in the continent
for their possible use in traditional medicine (Nneamaka, 1991). Research into traditional
plants and herbs received further boost due to the increasing resistance to many orthodox
medicine and thus a search for new organic molecules of plants with antimicrobial
properties (Sofowora, 1993). The use of plant extracts and phyto-products is gaining
 AASCIT Journal of Bioscience 2017; 3(4): 29-34
attention due to their availability, cost effectiveness, proven
nature of specificity, biodegradability, low toxicity, and
minimum residual toxicity in the ecosystem (Ogbo and
Oyibo, 2008). A lot of work has been carried out to prove
that several plant species possess antifungal and antibacterial
properties (Ficker et al., 2003; Erdogrul, 2002; Maji et al.,
2005). Persea americana (avocado pear) is one of such
plants. Persea americana is one of the 150 varieties of
avocado pear (Pacific Health Information, 2005). The tree is
widely cultivated in tropical and subtropical area, with a
height of about 80 feet, leathery, evergreen leaves, the
flowers are rarely unisexual. The seed of Persea americana
has a diverse application in ethnomedicine, ranging from
treatment for diarrhea, dysentery, toothache, intestinal
parasites to the area of skin treatment and beautification
(Pamplora and Roger, 1999). The seeds are rich in tannins
and carotenoids and tocopherols from the fruit were shown to
inhibit the in-vitro growth of prostate cancer cell lines (Lu et
al., 2005) and "persin" from avocado leaves was shown to
have antifungal properties and to be toxic to silkworms
(Oelrichs et al., 1995). The effect of Persea americana
extract was evaluated on in-vitro rat lymphocyte proliferation
(Gomez-Flores et al., 2008). Antioxidant activity and
phenolic content of seeds of avocado pear was found to be
greater than 70% (Soong and Barlow, 2004). The various
parts of Persea americana have been evaluated for various
biological activities. The leaf extract has been reported to
possess analgesic and anti-inflammatory activity (Adeyemi et
al., 2002), hypoglycemic activity (Antia et al., 2005),
hypoglycemic and hypocholesterolemic potential
(Bartholomew et al., 2007), body weight and liver lipids
effects (Brai et al., 2007), antimicrobial activity (Gomez-
Flores et al., 2008), anticonvulsant activity (Ojewole and
Amabeoku, 2006), hypotensive activity (Adeboye, 1999),
antiulcer activity and vaso-relaxant activity (Antia et al.,
2005). Persea americana root and stem bark extracts also
possess antibacterial activity against Staphylococcus aureus,
Escherichia coli, Pseudomonas aeruginosa, Serratia
marcescens, Enterobacter aerogenes and Klebseilla
pneumonia which could cause serious infections (Iqbal and
Arina, 2001). The present study was carried out to evaluate
the phytochemical and antibacterial activity of the leaf
extract of Persea americana against clinical bacterial isolates
that have shown some form of resistance of common
antibiotics.
2. Materials and Methods
2.1. Plant Materials
Persia americana leaves were obtained from the tree. The
leaves were air dried, grounded using a blender. The
powdered leaf was kept in bottle container until required.
2.2. Preparation of Crude Extracts
The method of Idris et al. (2009) was used. Fifty gram
30
(50g) of the grinded plant material was soaked in 250ml each
of distilled water and ethanol for 24 hours. The extract was
filtered through a sieve to remove debris. The filtrate was
then filtered through filter paper. The final filtrate was
evaporated in a water bath at 40°C to get the crude extract.
The crude aqueous and ethanol extract was stored at 4°C
until required. This was used for phytochemical and
antimicrobial analysis.
2.3. Preparation of Concentration of Plant
Extract
One gram (1g) each of both ethanol and aqueous extract
was added to 5ml of ethanol and distilled water respectively
to give a concentration of 200mg/ml. Other concentrations of
100, 50, 25 and 12.5mg/ml were prepared by double dilution
method as described by Iqbal and Arina (2001).
2.4. Phytochemical Screening of Extract
Phytochemical parameters of glycosides, steroids, flavonoids,
tannins, alkaloids, proteins, saponins, quinines and sugars of
the Persia americana were determined by standard methods.
2.4.1. Test for Glycosides
A 25ml of dilute H2SO4 was added to 5ml of plant extract
in a 100 ml flask. It was boiled (15 min), cooled and
neutralized with 10% NaOH. The fehling solution A and B (5
ml) was added to the neutralized solution and a brick red
precipitate of reducing sugars indicates the presence of
glycosides.
2.4.2. Test for Steroids
One gram of the test substance (plant extracts) was
dissolved in a few drops of acetic acid. It was gently warmed
and cooled under the tap water and a drop of concentrated
sulphuric acid was added along the sides of the test tube.
Appearance of green colour indicates the presence of Steroids.
2.4.3. Test for Tannins
The substance (extracts) was mixed with basic lead acetate
solution. Absence of formation of white precipitate indicated
the absence of Tannins.
2.4.4. Test for Alkaloids
Test substance (plant extracts powder) was shaken with
few drops of 2N HCL. An aqueous layer formed which was
decanted and one or two drops of Mayer’s reagent added.
Formation of white turbidity or precipitate indicates the
presence of alkaloids
2.4.5. Test for Flavonoids
Shinado’s test: To the substance (extracts) in alcohol, few
magnesium turnings and few drops of concentrated
hydrochloric acid were added and boiled for five minutes.
Red coloration shows the presence of Flavonoids.
2.4.6. Test for Saponins
The substance (extracts) shaken with water, foamy lather
formation indicates the presence of saponins.
31 Evbuomwan Lucky and Inetianbor Jonathan: Antibacterial Activity of Persia americana Leaf Extracts
Against Multidrug Resistant Bacterial Isolates
2.4.7. Test for Quinones
To the test substance, sodium hydroxide was added. Blue
green or red color indicates the presence of Quinone.
2.4.8. Test for Protein
To the test solution the Biuret Reagent is added. The blue
reagent turns violet in the presence of proteins.
2.4.9. Test for Reducing Sugars
The substance was mixed with equal volume of Fehling’s
A and B solutions, heated in water bath. Formation of red
colour indicates of the presence of sugar.
2.5. Test Microorganisms
Two Gram positive (Staphylococcus aureus and
Streptococcus pneumoniae) and three Gram negative
(Escherichia coli, Pseudomonas aeruginosa and Klebsiella
pneumoniae) were used in this study. The microorganisms
were obtained from the Microbiology Laboratory stocks in
University of Benin Teaching hospital. The bacterial strains
were grown in nutrient agar (NA) plates at 37°C.
2.6. Antibiotics Susceptibility Testing of the
Test Organism
Antimicrobial disc tests of the test organisms were
performed according to the recommendations of the National
Committee for Clinical Laboratory Standards (NCCLS, 2000)
using the following antibiotic discs; ampiclox (30ug), zinnacef
(20ug), amoxicillin (30ug), rocephin (25ug), ciprofloxacin
(l0ug), streptomycin (30ug), augmentin (20ug), sparfloxacin
(30ug), erythromycin (l0ug), gentamycin (10ug), septrin
(30ug), chloramphenicol (25ug), perfloxacin (l0ug), and
ofloxacin (30ug) and antibiotics resistance was interpreted by
diameter of inhibition zones around the antibiotic discs.
2.7. Antibacterial Susceptibility Testing of
the Extracts with Test the Organisms
The inocula were prepared by inoculating the test
organisms in nutrient broth and they were incubated for 24
hours at 37°C. The cultures were diluted to 0.5 McFarland
turbidity standard after the incubation. 0.2 milliliter of the
cultures was further diluted in normal saline and were
inoculated onto solidified nutrient agar using glass rod by
spreading technique. The ability of the various extracts to
inhibit the growth of the clinical test organisms was
determined using the agar well technique. The inoculated
nutrients agar plates were allowed to dry. After which, wells
were bored on the surface of inoculated agar plates using
4mm cork borer. 0.2 ml of the different concentration of each
extracts were transferred into the well using Pasteur pipette.
The wells were sufficiently spaced to prevent the resulting
zones of inhibition from overlapping. The plates were
incubated at 37°C for 24hours. The experiment was
performed in triplicate and the resulting zones of inhibition
were recorded as mean ± standard error
2.8. Determination of Minimum Inhibitory
Concentration (MIC) and Minimum
Bactericidal Concentration (MBC)
The minimum inhibitory concentration (MIC) of the
methanol extracts was determined for each of the test organisms
in triplicates at varying concentrations of 200, 100, 50, 25, 12.5,
6.25 and 3.125 mg/ml). 1 ml of nutrient broth was added and
then a loopful of the test organism previously diluted to 0.5
McFarland turbidity standard was introduced to the tubes. A
tube containing nutrient broth only was seeded with the test
organism to serve as control. All the tubes were then incubated
at 37°C for 24 hours and then examined for growth by observing
for turbidity. The minimum bactericidal concentration (MBC) of
the plant extract on the clinical bacterial isolates was carried out
according to Ajaiyeoba et al. (2003). Briefly, 1 ml bacterial
culture was pipetted from the mixture obtained in the
determination of MIC tubes which did not show any growth and
were sub-cultured onto nutrient agar and incubated at 37°C for
24 hours. After incubation the concentration at which there was
no single colony of bacteria was taken as MBC
3. Results
In order to determine the phytochemical parameter or
constituents of Persia americana, phytochemical analysis
was carried using aqueous and ethanol extract of the Persia
americana leaves. Table 1 shows the phytochemical analysis
of Persia americana. Similar phytochemicals parameters
were obtained from the two extract (aqueous and ethanol).
Tannins was however not detected from the two extracts. In
order to confirm the antibiotic resistance pattern or strength
of the test organisms obtained from the university of Benin
teaching hospital, antibiotic susceptibility testing was carried
(table 2) and the percentage of resistance among the sample
organisms was also tested (figure 1). Table 3 shows the mean
antibacterial activity of Persia americana at various
concentrations of ethanolic extract. The antibacterial activity
of Persia americana in aqueous was also carried out in
aqueous extract as shown in table 4. The zones of inhibition
shown the degree of susceptibility measured in millimeters
(mm). Table 5 show the MIC and MBC of both ethanol and
aqueous extract of Persia americana
Table 1. Phytochemical analysis of Persia americana with ethanol and
aqueous extracts.
Phytochemicals Solvents
Ethanol Aqueous
Flavonoids + +
Tannins - -
Cardiac glycosides + +
Reducing sugars + +
Terpenoids + +
Saponins + +
Proteins + +
Alkaloids + +
Steroids + +
Key: + = present, - = absent
 AASCIT Journal of Bioscience 2017; 3(4): 29-34 32

Table 2. Antibiotic susceptibility testing pattern with the test organism (Positive control).

Gram +ve CPX St SXT E PEF CN APX Z AM Ro

S. aureus S R R S S S R R R S
S. pneumonia S R R R R R R R R S
Gram –ve CH SP AU OFX SXT PEF AM St CN CPX
E. coli S S S S R S R S R S
P. aeruginosa R R S S R S R R S S
K. pneumonia S S S S R S S R S S

The antibiotic susceptibility testing pattern of the test organism show that S. pneumonia was the most resistant test organism
to conventional antibiotics tested while K. pneumonia was the most susceptible organism to the antibiotics used. This positive
control gave an insight into the susceptibility and sensitivities of the test organisms against conventional antibiotics. KEY:
CPX = Ciprofloxacin, Ro = Rocephin, St = Streptomycin, AU = Augmentin, SXT = Septrin, SP = Sparfloxacin, E =
Erythromycin, CH = Chloramphenicol, PEF = Pefloxacin, CPX = ciprofloxacin, CN= Gentamicin, APX = Apmpiclox, AM=
Amoxacillin and, Z = Zinnacef, OFX = ofloxacin

Figure 1. Bar chart of the percentage of antibiotic resistance with the test organisms. The resistance (%) of the different test organisms (bacteria) to the
antibiotics revealed that S. pneumonia (80%) was the most resistance organism followed by S. aureus and P. aeruginosa (50%), E. coli (30%) and K.
pneumonia (20%).

Table 3. Mean antibacterial activity of Persia americana at various concentrations of the ethanol extract (zones of inhibition were measured in millimeters).

Test organisms Concentrations

200mg/ml 100mg/ml 50mg/ml 25mg/ml 12.5 mg/ml 6.25 mg/ml 3.125 mg/ml
S. aureus 21.0±1.0 18.5±0.5 17.5±2.5 15.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
E. coli 26.0±1.0 18.5±1.5 18.0±3.0 15.0±5.0 0.0±0.0 0.0±0.0 0.0±0.0
S. pneumonia 15.0±0.0 11.0±1.0 10.0±1.0 9.5±0.5 0.0±0.0 0.0±0.0 0.0±0.0
K. pneumonia 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
P. aeruginosa 13.5±1.5 12.5±1.5 7.5±1.5 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0

The antibiotics activity of Persia americana at various concentration of the ethanol extract show that the mean highest zone
of inhibition in millimeters was with E. coli (26.0±1.0) at 200mg/ml concentration while the least zone of inhibition was with
K. pneumonia (0.0±0.0) at all the concentration. There was however no activity at 12.5mg/ml, 6.25mg/ml and 3.125mg/ml
concentrations against the test organisms

Table 4. Mean antibacterial activity of Persia americana at various concentrations of aqueous extract (zones of inhibition are measured in millimeters).

Test organisms Concentrations

200mg/ml 100mg/ml 50mg/ml 25mg/ml 12.5mg/ml 6.25mg/ml 3.125mg/ml
S. aureus 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
E. coli 11.0±0.0 8.5±3.5 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
S. pneumonia 15.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
K. pneumonia 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
P. aeruginosa 20.0±5.0 17.5±2.5 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0
33 Evbuomwan Lucky and Inetianbor Jonathan: Antibacterial Activity of Persia americana Leaf Extracts
Against Multidrug Resistant Bacterial Isolates
The antibiotics activity of Persia americana at various
concentration of the aqueous extract show that the mean
highest zone of inhibition in millimeters was with P.
aeruginosa (20.0±5.0) at 200mg/ml concentration while the
least zone of inhibition was with S. aureus and K.
pneumoniae (0.0±0.0) at all the concentration. There was
however no activity at 12.5mg/ml, 6.25mg/ml and
3.125mg/ml concentrations against the test organism.
Table 5. MIC and MBC of ethanol and aqueous extract of Persia americana.
MIC(mg/ml) MBC (mg/ml)
Test organisms
Ethanol Aqueous Ethanol Aqueous
S. aureus 25 - 100 - be highly resistant to the plant at all concentrations. This
E. coli 25 100 100 200
S. pneumonia 25 200 50 -
K. pneumonia - - - -
P. aeruginosa 50 50 50 200
In the ethanolic extract, 25mg/ml was the lowest
concentration that inhibited the growth of Staphylococcus
aureus, Escherichia coli and Streptococcus pneumoniae;
While 50 mg/ml was the MIC for Pseudomonas aeruginosa.
Minimum bactericidal concentration of 50mg/ml was
observed against Streptococcus pneuoniae and Pseudomonas
aeruginosa while MBC of 100mg/ml was observed against
Staphylococcus aureus and Escherichia coli. In the aqueous
extract, Pseudomonas aeruginosa has the lowest MIC value
of 50mg/ml while Streptococcus pneumoniae had the highest
MIC of 200mg/ml. Minimum bactericidal concentration of
200mg/ml was observed against Escherichia coli and
Pseudomonas aeruginosa
4. Discussion
The phytochemical screening of Persia americana
revealed the presence of flavonoids, saponins, steroids,
reducing sugar, cardiac glycosides, alkaloids, proteins and
terpenoids in both ethanolic and aqueous extracts while
tannin was absent in both extracts. Flavonoids are known to
be synthesized by plants in response to microbial attack.
Hence, it should not be surprising that they have been found
to be effective antimicrobial substances against a wide array
of microorganisms, when tested in-vitro. Their activity is
probably due to their ability to react with extracellular and
soluble proteins and to complex with bacterial cell walls
leading to the death of the bacteria (Cowan, 2002; Idris et al.,
2009). The spectra of antimicrobial activities displayed by
these extracts could perhaps be explained by the presence of
flavonoids, saponins and steroids. The purified components
may even have more potency with respect to inhibition of
microbes. The result of the present study signifies the
potential of P. americana as a source of therapeutic agents,
which may provide leads in the ongoing search for
antimicrobial agents from plants. Further studies on the
phytoconstituents and purification of individual groups of
bioactive components can reveal the exact potential(s) of the
plant to inhibit several pathogenic microbes. The
antibacterial activity of the extract was observed to be
dependent on the type of solvent for extraction and also on
the concentration of the crude extract used. The ethanolic
extract was found to be more active than the aqueous extract.
At the lowest concentration of 25mg/ml, the ethanol extract
had antibacterial activity against S. aureus, E. coli and S.
pneumoniae with zones of inhibition of 15.0±0.0, 15.0±5.0
and 9.5±0.5mm respectively. The ethanol extract was more
potent at higher concentrations with higher inhibition zones
of 26.0±1.0mm and 21.0±1.0mm against E. coli and S.
aureus respectively. Klebsiella pneumoniae was observed to
study shows that ethanol was able to extract more of the
bioactive phytoconstiuents from the plant. The antibacterial
activity of the aqueous extract was also dependent on the
concentration of the extract. S. aureus and K. pneumoniae
were completely resistant to all concentrations of the aqueous
extract. E. coli was susceptible to the extract at 200mg/ml
and 100mg/ml with zones of inhibition of 11.0±0.0mm and
8.5±3.5mm respectively. S. pneumoniae was only susceptible
at 200mg/ml with inhibition zone of 15.0±0.0mm while P.
aeruginosa was sensitive at 200 and 100mg/ml, with
inhibition zones of 20.0±5.0mm and 17.5±2.5mm
respectively. It can be shown from this study that the aqueous
medium did not extract much of the bioactive
phytoconstituents, hence its reduced antibacterial activity
against the test organisms in vitro. This might however be
different if an in-vivo study is carried out. Minimum
inhibitory concentration of 25mg/ml was recorded against S.
aureus, E. coli and S. pneumoniae in the ethanol extract of
the plant. The MIC values in the aqueous extract were higher,
ranging from 50mg/ml to 200mg/ml. Minimum bactericidal
activity ranged from 50mg/ml to 100mg/ml in the ethanol
extract and 200mg/ml against P. aeruginosa and E. coli in
the aqueous extract. This infers that the therapeutic activity
of P. americana could be harnessed by using ethanol as
solvent for extraction. Antibiotic sensitivity pattern of the test
bacterial strain revealed varying degree of resistance to
conventionally used antibiotics. The most resistant bacterium
was S. pneumoniae (80%) while the most sensitive was K.
pneumoniae (20% resistance). All the organisms were
observed to be multiple drug resistance. This attributes could
be due to plasmid borne or chromosomally mediated resistant
genes in the test isolates. In this study, extract of Persia
americana has been shown be comparatively more
antibacterial than conventionally used antibiotics.
5. Conclusion
This work has shown that Persea americana leaf extract
has potent antibacterial activity which is dependent on the
solvent used for the extraction. Many of the bacterial test
organisms which were resistant to conventional antibiotics
were observed to be sensitive to the plant extract. Ethanol
 AASCIT Journal of Bioscience 2017; 3(4): 29-34
extract was more potent, showing its potential applicability in
extraction and purification of phytochemicals from plants.
Persea americana should be seen as a potential source of
therapeutic agents and used as an alternative to conventional
antibiotics. However, more research work on its toxicity level,
possible synergistic or antagonistic interaction with other
plants or drugs, is needed to consolidate its usage.
Conflicting Interests
The authors declare that there is no conflict of interest.
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