Ethnobotanical
Leaflets 12: 1035-42. 2008.
Antibacterial Effect
of some Plant Extracts on Selected Enterobacteriaceae
*I.O. Sule and T.O. Agbabiaka Department of
Microbiology, *Corresponding E-Mail:
ismailaolawales@yahoo.co.uk Issued ABSTRACT Vernonia amygdalina (Bitterleaf), Eucalyptus citriodora
(Eucalypt) and Phyllanthus amarus (Schum) were investigated
for their antibacterial properties against pure cultures of clinical isolates
of Escherichia coli, Klebsiella sp., Salmonella
sp. and Shigella sp. The isolates
were obtained from dept of Medical Microbiology and Parasitology
of the Water
extracts of vernonia amygdalina
(Bitterleaf) and Schum
(Phyllanthus amarus)
were not effective on majority of the test organisms. Klebsiella
sp. was not inhibited by the water extracts at the test concentrations.
The Ethanolic extracts of Eucalyptus citriodora (Eucalypt) were most effective on all the
test organisms. The least and the most susceptible organisms to the extracts
were Shigella sp. and E. coli
respectively. The results of this study suggest the possibility of using the ethanolic extracts of these plants in treating diseases
caused by the test organisms. Key Words: Plant extracts, Antibacterial effect,
Inhibition, Enterobacteriaceae. INTRODUCTION Antibacterial
activity is the ability of a substance to inhibit or kill bacterial cells.
Different types of antibiotics and chemotherapeutic agents are being used in
the treatment of one form of disease or the other. Most of these antibiotics
were originally derived from micro-organisms while the chemotherapeutic
agents are from plants. However, nowadays these antibiotics and
chemotherapeutic agents are obtained by various synthetic processes (Reiner, 1984). Most countries in The
leaves, stems, bark, roots etc of these plants are
being used by the local populace and people with thin income for incurring
different types of ailments because of the inadequate medical facilities
across the nook and cranny of these countries. Some of the plants which are being used
medicinally in Enterobacteriaceae is one of the most widely studied family of bacteria. The members of this family are gram
negative, rod shaped, non-sporulating and
facultative anaerobes. Some members of this family are able to ferment
lactose with the production of acid and gas. These are called coliforms. Examples of coliforms
are E.coli, Klebsiella,
Enterobacter, Citrobacter
etc. However, others do not ferment lactose and includes Shigella sp. and Salmonella sp. Most
of the members of this family enterobacteriaceae cause
infection in their host. Infection
by Salmonella sp. is contacted by ingesting large numbers of viable
bacteria in faecally contaminated food or water.
The symptoms include diarrhoea and abdominal cramps
with nausea and vomiting. Good sanitation and avoiding sewage contamination
of water by domestic animals and fowl help to reduce the incident of
Salmonella infections. Similarly,
the symptoms of bacillary dysentery caused by Shigella
dysenteriae are initial fever and abdominal
cramps, then diarrhoea with profuse bloody stools.
High temperature and vomiting may also occur. The control of shigellosis
requires disruption of the anal-oral route of transmission by means of good
sanitary practice. The
natural habitat of E. coli is the alimentary tract of man and warm
blooded animals; it is one of the most abundant of the intestinal flora.
Various strains of E. coli have been implicated in the outbreaks of diarrhoeal illness and their routes of transmission have
been traced to sewage contaminated drinking water. These strains of E.
coli include: Enteropathogenic strains of E.
coli (EPEC); Enteroinvasive E. coli (EIEC) and Enterotoxigenic E. coli (ETEC). They cause diarrhoea
in children and/ or adults (Sterritt and Lester,
1988). The
local populaces rely heavily on most of these medicinal plants for treating
various diseases caused by some of these agents. The plants for this study Vernonia amygdalina (Bitterleaf), Eucalyptus citriodora
(Eucalypt tree) and Phyllanthus amarus (Schum) have also
been used for treatments locally (Akujobi et
al., 2006). Vernonia amygdalina (Bitterleaf) belongs to the family of compositae
(Keay, 1989). It is particularly abundant in
grassland throughout the tropics and warmer regions. It is well known as a
source of chewing stick for their bitter taste. It is a popular leafy
vegetable especially among the Ibos of Eastern Nigeria. Eucalyptus
is a lemon scented gum tree. Industrially, Eucalyptus oil is obtained by a process
of distillation from the fresh leaves of this plant and have
been used in treating colds, coughs, catarrh, cuts, scratches etc (B.P.
1993). Schum (Phyllanthus amarus) is an herbaceous plant and have equally been
used to cure various ailments. Therefore,
this study was primarily undertaken to confirm the acclaimed antibacterial
properties of Vernonia amygdalina, Eucalyptus citriodora
and Phyllanthus amarus
based on their ethnomedical uses in MATERIALS
Collection
and Identification of Plant Materials Eucalyptus
citriodora (Eucalypt) and Phyllanthus
amarus (Schum) were
collected at the permanent site of Test
Organisms The test organisms for this study
were members of the family Enterobacteriaceae,
namely: Escherichia coli, Klebsiella
sp., Salmonella sp. and Shigella sp. The
pure clinical isolates were obtained from the department of Medical
Microbiology and Parasitology, Sample
Preparation and Extraction
Each
of the plant material was grinded and 10g of it was added to 100ml of
distilled water or 70%w/v ethanol in order to obtain water or Ethanolic extract (100mg/ml). This crude extraction was
done at room temperature for 24 hours. Muslin cloth was then used to filter
the plant residues and the filtrate thus obtained was further purified by
filtration through Whatman No 1 filter paper (Atata et al., 2003). This stock solution of
extract was sterilized by filtration through Millipore membrane filter of
0.45μm pore-size (Ronald, 1995). The sterile extract obtained was stored
in sterile capped bottles and refrigerated at 40c until when
required for use. Sterility
Proofing of the Extracts
The
extract was tested for sterility after Millipore filtration by introducing
2ml of this supposed sterile extract into 10ml of sterile nutrient broth.
Incubation was done at 370c for 24hours. A sterile extract was
indicated by absence of turbidity or clearness of the broth after the incubation
period (Ronald, 1995). Standardization of the Bacterial Cell Suspension Five
colonies of each test organism were picked into sterile testtube
containing sterile nutrient broth and incubated at 370c for 24
hours. The turbidity produced by this organism was adjusted and used to match
the turbidity (opacity) standard prepared as described by Monica (1984). Determination of Minimum
Inhibitory Concentration (MIC) of the extracts on the test organisms The
initial concentration of the plant extract (100mg/ml) was diluted using
double fold serial dilution by transferring 5ml of the sterile plant extract
(stock solution) into 5ml of sterile Nutrient broth to obtain 50mg/ml
concentration. The above process was repeated several times to obtain other
dilutions: 25mg/ml, 12.5mg/ml, 6.25mg/ml and finally 3.125mg/ml (Ibekwe et al., 2001). Having obtained the
different concentrations of the extracts, each concentration was inoculated
with 0.1ml of the standardized bacterial cell suspension and incubation was
done at 370c for 24 hours. The growth of the inoculum
in the broth is indicated by turbidity or cloudiness of the broth and the
lowest concentration of the extract which inhibited the growth of the test
organism was taken as the Minimum Inhibitory
Concentration (MIC). Negative controls were set up as follows: Nutrient broth
only; Nutrient broth and sterile plant extract; and finally positive control
containing Nutrient broth, and a test organism. Determination
of Zones of Inhibition
Fifteen
millimetre (15ml) of
sterile Nutrient agar was poured into each sterile petridish
of equal size and allowed to solidify. The surface of this sterile Nutrient
agar plate was streaked with the pure culture of the standardized bacterial
cell suspension. A corkborer, (8mmin diameter) was
sterilized by flaming and used to create ditch at the center of the plate.
The hole so created was then filled with the plant extract. The plates were
allowed to stand for one hour for pre-diffusion of the extracts (Esimone et al., 1998) and incubation was done at
370c for 24hours. At the end of the incubation period, the
diameter of zone of inhibition was measured in millimetre
(Hugo and Russel, 1996). Table 1. Determination of Minimum Inhibitory Concentration
(MIC) of the water extracts on the test organisms.
-, No inhibition at the concentrations used. Table 2. Determination of Minimum Inhibitory
Concentration (MIC) of the Ethanolic Extracts on
the Test Organisms.
Table 3. Diameters of Zones of Inhibition of
the Water extracts (100mg/ml) on the test organisms.
NI: No inhibition at the concentration
used. Table 4. Diameters of zones of inhibition of
the ethanolic extracts (100mg/ml) on the test
organisms.
NI: No inhibition at the concentration
used. RESULTS The results of the Minimum Inhibitory
Concentration (MIC) showed that majority of the test organisms were not
inhibited by the water extracts at the test concentration used. However, the
water extracts of Eucalypt and Schum had MIC of
50.0mg/ml on E. coli and Salmonella sp. respectively (Table 1).
In
contrast, all the ethanolic extracts exerted
inhibitory effect on the test organisms to different extent (Table 2) Salmonella
sp. was most susceptible to the ethanolic
extract of Bitterleaf, this was followed by Klebsiella sp. (MIC 12.5mg/ml) and E.
coli (MIC 25.0mg/ml). Shigella sp. was
the least susceptible (MIC 50.0 mg/ml). The most susceptible test organism to
the ethanolic extracts of Eucalypt and Schum was Salmonella sp. whereas the other test
organisms were inhibited to the same extent (MIC 50.0mg/ml). The
results obtained in the Agar diffusion plates followed the same trend with
what was obtained in the Minimum Inhibitory tests. The water extracts of Schum was not inhibitory on all the test organisms. Similarly,
the water extract of Bitterleaf only inhibit Salmonella
sp. where it created a diameter of zone of inhibition of 3.0mm. Eucalypt
inhibited all the test organisms to different extents except Klebsiella sp. where no inhibition was
observed (Table 3) at the concentration used. The
ethanolic extracts of the plants inhibit the test
organisms to different degrees except Bitterleaf
which failed to show inhibitory effect on Shigella
sp. at the concentration used. In all cases, the ethanolic
extracts of Eucalypt had the most inhibitory effect
on the test organisms and this was followed by Schum
(Table 4). DISCUSSION The
results of the inhibitory effects of the water extracts showed that it is
less effective on the test organisms than the ethanolic
extracts (Tables 1-4). In
the broth dilution tubes for the MIC, the water extract of Bitterleaf was not inhibitory on all the test organisms.
Eucalypt and Schum followed similar trend being
inhibitory on only 25% of the test organisms. This implies that the crude
water extracts of these plants could not be suitable (at 6.25 - 50.0mg/ml) in
tackling diseases caused by these test organisms as it is sometimes used by
the local populace. If water must be used for extraction, Eucalypt will be
most suitable (Tables 1 and 3). Investigators
in the past had also clearly shown that ethanolic
extracts were more effective than water extract (Ibekwe
et al., 2001; and Dutta 1993). They have
attributed this observation to the high volatility of ethanol which tends to
extract more active compound from the sample than water. Hence, these studies
followed similar trends. Klebsiella sp. was not inhibited in the
water extracts (Tables 1 and 3). However, it was inhibited by the ethanolic extracts. This may be due to the fact that this
organism produces capsule which would not be readily dissolved in water. In
all cases where possible, the ethanolic extracts of
these plants should be used at a concentration up to 100mg/ml (Table 4) so as
to give a better treatment margin than the maximum 50.0mg/ml obtained in the
MIC tests (Table 2). From
the Agar diffusion plates, the ethanolic extracts
of Eucalypt had the greatest antibacterial effects and this was followed by Schum. Bitterleaf had the least
antibacterial effect on all the test organisms. It however, failed to inhibit
Shigella sp. at the concentration
used. Therefore, the order of ease of susceptibility of the test organisms to
the different Ethanolic extracts were: E. coli; Klebsiella sp., Salmonella sp. and finally Shigella sp. CONCLUSION This
investigation revealed that the water extract of Eucalypt exert appreciable
antibacterial effect on all the test organisms except Klebisiella
sp. and that the water extract of Bitterleaf is
the least effective on all the test organisms. Furthermore, this study also
revealed that the ethanolic extract of Eucalypt had
the highest antibacterial effect on all the test organisms, followed by Schum and finally Bitterleaf.
Eucalypt is therefore recommended for usage by the local populace because of
its significant antibacterial effect as revealed by this study. REFERENCES Akujobi C.O., Ogbulie
J.N. and Uchegbu U.N. (2006). Antibacterial
activities and preliminary phytochemical screening of Vernonia amygdalina and Citrus aurantifolia. Nig. Journal of
Microbiol. 20 (11). pp. 649-654. Atata R.F., Sani
A. and Ajewole S.M. (2003). Effect of Stem Bark
extracts of Enantia chloranta
on some Clinical Isolates. Nig. Society for Experimental Biology. 15
(2): 84-92. British Pharmacopeia (1993). Volume I, International Edition,
HMSO Publications Centre, Dutta A.C. (1993). Botany for Degree
Students. 5th edition. Ebana F.U.B., Madunagu
B.E. and Etok Esimone C.O., Adikwu
M.U. and Okonta J.M. (1998). Preliminary
antimicrobial screening of the ethanolic extract
from the lichen Usnea subfloridans
(L). Journal of Pharmaceutical Research and Development. 3(2): 99-101. Hugo W.B. and Russell A.D.
(1992). Pharmaceutical Microbiology. 5th Ed. Blackwell
Scientific Publications Ibekwe V.I., Nnanyere
N.F. and Akujobi C.O. (2001). Studies on
Antibacterial activity and phytochemical qualities
of extracts of orange peels. Int. J. Environ., Health and Human Development. 2(1):41-46.
Keay R.W.J. (1989). Trees of Monica C. (1984). Medical
Laboratory Manual for Tropical Countries, Vol. 2. Microbiology.
Butterworth and Co (Publishers) Ltd. Borough Green, Reiner R. (1984). Antibiotic: An
Introduction. New Horn Publishing Co. Ronald M.A. (1995). Micro-organisms
in our World. Mosby Year Book, Inc. Sterritt R.M. and Lester J.N. (1988). Microbiology
for Environmental and Public Health. Engineers E & F. N. Spon Ltd., |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||