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Ethnobotanical Leaflets 13: 522-31. 2009. Tradomedical Values of
Cotton Leaf Plus Lemon Juice Against Clinical Bacterial Isolates *I.O. Sule,
T.O. Agbabiaka and G.P. Oyeyiola Department of Microbiology, *Corresponding
Editor E-Mail: ismailaolawales@yahoo.co.uk Issued Abstract
The antibacterial
activity of the water and ethanolic extracts of
cotton leaf (Gossypium spp.) plus lemon juice (Citrus limon)
were tested against pure clinical isolates of Salmonella sp., Shigella sp., E. coli
and Klebsiella sp. Cotton leaf is
normally used in conjunction with lemon juice by the local populace in
Nigeria for the treatment of enteric infections. Decoction method was used
for the extraction of the active components from the plant in order to
simulate the traditional method of extraction. The Minimum Inhibitory
Concentration (MIC) and the diameters of zones of inhibition were determined
by broth dilution and Agar diffusion methods respectively. The ethanolic extracts are more effective than the water
extracts on the test organisms. The MIC of the water and ethanolic
extracts ranged between 1.25 -5.0 w/v on the test organisms. Similarly, the
average diameter of zones of inhibition of the water extracts on the test
organisms ranged between 3.0 to 13.0mm while that of the ethanolic
extracts ranged between 12.0 to 21.0mm. The results of this study showed that
E. coli was
the most susceptible followed by Klebsiella sp.,
then Salmonella sp., and finally Shigella sp. at the concentrations used for
both water and ethanolic extracts. This observation
thereby justifies the traditional uses of these plant extracts among the
Nigerian local populace for the treatment of some enteric infections such as
dysentery and diarrhoea. Key words:
Cotton leaf plus lemon extracts, antibacterial activity, inhibition, clinical
bacterial isolates. Introduction
Cotton (Gossypium
spp.) is a tropical plant belonging to the
family Malvaceae while lemon (Citrus limon) which is a tree crop belongs to the family Rutaceae (Ghazanfar, 1989).
Cotton is widely cultivated in the Northern part of Historical data has shown that
many chemotherapeutic drugs or compounds known today were formally derived
from plants (Reiner, 1984). For instance, the first
importation of Cinchona to Scientists in most of these third
world countries where a large proportion of their population still uses these
medicinal plants are saddled with the responsibilities of trying to
authenticate the veracity of these supposed curative or medicinal effects.
Many Nigerian scientists from the various parts of the country have risen up
to these challenges and have done considerable research works on many of
these local medicinal plants. For instance, Akujobi
et al. (2006) worked on the antibacterial activities and phytochemical screening of Vernonia
amygdalina and Citrus aurantifolia.
Similarly, Kaufman et al. (1989) has shown that Citrus spp contains cardiac glycosides, saponins,
tannins and alkaloids. There is little scientific
research on the leaves of cotton plant (Gossypium
spp.) but some investigations have been done on
the different varieties of Citrus. However, in the tradomedical
practices in Materials and Methods
Plant Materials collection and
Identification
The leaves of cotton plants were
obtained from a farm at Osere district in Preparation of Plant Extracts
The plucked leaves of cotton plant
were washed and crushed by means of sterile mortar and pestle. The aqueous
extract was prepared by weighing 10g of the grinded cotton leaf and soaking
it in a mixture of 50ml each of the squeezed lemon juice and distilled water.
Similarly, another 10g of the crushed cotton leaf was
soaked in a mixture of 50ml each of the squeezed lemon juice and 70% v/v
ethanol. Soaking in both cases were done at room temperature for a period of
24hours. At the end of this period, the extract was shaken and passed through
muslin cloth in order to remove the residue. This residue free extract
(filtrate) was then further filtered through Whatman
No.1 filter paper for better clarity of the solution. Sterilization of the Plant
Extracts
These aqueous and ethanolic extracts (filtrates) obtained above were
rendered sterile by aseptic filtration through Millipore filter of size
0.45μm with the aid of Millipore filtration apparatus connected to a
vacuum pump (Ronald, 1995). Sterility Proofing of the
Extracts
This was done by introducing 2ml
of the sterile filtrate obtained above into 10ml of sterile Tryptone soy broth. Incubation was done at 370C
for 24hours. At the end of this period, the broth was observed for the
absence of turbidity as an indication of sterility (Atata
et al., 2003; Sule and Agbabiaka,
2008). Collection
of test Organisms Pure clinical isolates of Salmonella
sp., Shigella sp., Escherichia coli
and Klebsiella sp. were collected from
the Department of Medical Microbiology and Parasitology
of the Standardization
of Culture Suspension Five colonies of each of the test
organisms were introduced into 5ml of sterile tryptone
soy broth and incubation was done for 24hours at 370C. The
turbidity (opacity) standard was prepared as described by Cheesbrough
(1984). This was then used to match the turbidity of the cell suspension of
an overnight broth culture of each test organism. Determination
of the Minimum Inhibitory Concentration (MIC) Double fold serial dilutions were
made using tryptone soy broth adopted from the
method of Ibekwe et al., (2001); Sule and Agbabiaka (2008). The
initial concentration of the plant extract was 10% w/v and from it the
following concentrations were obtained: 5.0% w/v, 2.5% w/v, 1.25% w/v, 0.625%
w/v and finally 0.312% w/v. The different dilutions of the broth above were
inoculated with 0.1ml of each pure and standardized bacterial suspension and
incubation was done at 370c for 24 hours. Growth of the inoculated
test organism was indicated by turbidity (Cloudiness) of the broth tubes
while clearness of the broth tube indicates absence of growth. The broth
dilution tube with the least concentration of extract where no growth was
observed was taken as the MIC. The control experiments were also set up as follows: Sterile
tryptone soy broth only; sterile tryptone soy broth and sterile extracts (Negative
controls) and finally sterile tryptone soy broth
inoculated with a test organism (positive control). Determination of Zones of
Inhibition
The diameter of zones of
inhibition of the extract on each of the test organism was determined using
agar diffusion method as described by Hugo and Russel
(1996). 15ml of the sterile molten Tryptone soy
Agar was poured into the sterile plate (8cm in diameter) and allowed to solidify.
The surface of the sterile, dried and solidified Tryptone
soy Agar plate was then streaked with the standardized cell suspension. Flamed Statistical
Analysis: t-test was used to determine
if there is any significant difference between the inhibition of the test
organisms in the water and ethanolic extracts for
both the broth dilution tubes(MIC) and agar diffusion plate (Bello and Ajayi,2000). Results
and Discussion Both the water and ethanolic extracts exhibited antibacterial activities
against the test organisms to different extents. However, in most cases the ethanolic extracts showed higher activities than the
water extracts. Based on the minimum inhibitory concentration (MIC) tests,
both Salmonella sp. and Shigella
sp. had the highest resistant in the water extract (5% w/v) followed by Klebsiella sp. (2.5% w/v) and lastly E. coli (1.25% w/v) has shown in Table 1. Similarly, in
the ethanolic extracts, Salmonella sp. and Shigella sp. were inhibited to the same
extent (5.0%w/v) in the MIC broth tubes and they were the least susceptible
organisms. Furthermore, it was observed that E. coli
and Klebsiella sp. has equal
resistant, their MIC being 1.25% w/v. They are more susceptible compared to Salmonella
sp. and Shigella sp. Three of the test organisms viz. Salmonella
sp., Shigella sp. and E. coli
had the same zone of inhibition in both the water and ethanolic
extracts. However, Klebsiella sp. showed
shift in susceptibility from 2.5% w/v in the water extract to 1.25% w/v in
the ethanolic extract of the MIC broth tubes. In
the Agar diffusion plates, the water extract at 10% w/v concentration created
a zone of inhibition of 3mm on Shigella
sp. This indicates the highest resistant. Its resistant was followed by
inhibitory zone of 10mm on Klebsiella
sp., 13mm on Salmonella sp. and the least resistant of 18mm on E.
coli. Similarly, in the ethanolic extracts using Agar diffusion method Klebsiella sp. was the most susceptible
organism with diameter of zone of inhibition of 21mm. The next susceptible
organism was E. coli with an inhibitory
diameter of 19mm and this was followed by Salmonella sp. that was
inhibited up to 13mm diameter. Shigella
sp. was the least susceptible among the test organisms with inhibitory
diameter of 12mm. The result of the
students’ t-test statistic showed that there was no significant difference
between the inhibition of the test organisms in both water and ethanolic extract at the concentrations of 0.312 –
5.0%w/v used in broth dilution tubes since the calculated value 3.2084 is
less than the critical table value 4.541.However,in the agar diffusion plates
at concentration of 10% w/v in both extracts there is significant difference
between the inhibition of the test organisms in the water and ethanolic extracts (calculated value 5.5468 greater than
the table value 4.541). The results from this study showed
that the leaf extract of Gossypium spp (Cotton plant) in conjunction with the juice of Citrus
limon (lemon fruit) have significant
antibacterial effects on all the clinical bacterial isolates used for this
investigation at the concentrations used. However, these antibacterial
activities were to different extents (Tables 1 to 3). Ethanol has a higher
volatility than water. Thus, it tends to extract more active compounds from
the leaves of the plants than water (Dutta, 1993).
This observation correlates with the works of Ibekwe
et al.(2001); Akujobi et al.(2004); Atata et al. (2003);
Sule and Agbabiaka
(2008). E. coli
was the most susceptible to the water extract among the test organisms
(Table1). It was also observed that both E. coli
and Klebsiella sp. were the most
susceptible in the ethanolic extract (Table 2). The
observation that Klebsiella sp. which
was less susceptible than E. coli in the
broth dilution tubes of the water extract having the same susceptibility with
it in the ethanolic extract could be explained by
the fact that Klebsiella sp. are
capsulated organism. Hence, the outer capsule of such organism would offer a
higher degree of being less soluble in water but are easily dissolved or solubilised in ethanol (Tables 1 and 2). Salmonella
sp. and Shigella sp. were the
most resistant among the test organisms in the broth dilution tube in both
the water and ethanolic extracts. In the Agar diffusion tests, the
concentration of the plant extract used was 10% w/v (Table 3). Salmonella
sp. was inhibited by both the water and ethanolic
extracts to the same extent (diameter of zones of inhibition being 13.0mm). Similarly,
E. coli showed almost the same inhibition in
both extracts. Furthermore, in the water extract the most resistant among the
test organisms was Shigella sp.
followed by Klebsiella sp. Salmonella sp.
and finally E.coli (most
susceptible). The most resistant among the test organisms in the ethanolic extract was still Shigella
sp. followed by Salmonella sp., E. coli
and Klebsiella sp. (most
susceptible). In this study, Decoction method
was used to extract the active components from the plant materials rather
than other efficient methods such as Soxhlet
apparatus. This was done in order to simulate the extraction’s method of the
local populace who use these plants for curing ailments. In
conclusion, this study suggests that the extracts of cotton leaf plus lemon
juice have potential inhibitory effects against the test organisms at the
concentrations used. Both the water and ethanolic
extracts showed significant inhibitory effects on all the test organisms
thereby providing alternative for those who dislike the use of ethanol for
religious or health reasons. However, it was observed that the ethanolic extract exert more or on a few occasion equal
inhibitory effect on all the test organisms. Hence, it is suggested that
ethanol should be used for extraction as much as possible. References Akujobi
C.O., Anyanwu B.N., Onyeze
G.O.C. and Ibekwe V.I. 2004. Antibacterial
Activities and Preliminary Phytochemical Screening
of four Medical plants. Journal of Applied Science 7(3):
4328-4338. Akujobi
C.O., Ogbulie J.N. and Uchegbu
U.N. 2006. Antibacterial activities and preliminary phytochemical
screening of Vernonia amygdalina
and Citrus aurantifolia.
Nigerian Journal of Microbiol. 20(11): 649-654. Atata, R. F.,
Sani A. and Ajewole S.M.
2003. Effect of stem bark extracts of Enantia chloranta on some clinical isolates. NISEB Journal
15(2): 84-92. Cheesbrough, M.
1984. Medical Laboratory Manual for Tropical Countries. Vol. 2: Microbiology.
Butterworth and Co. (Publishers) Ltd., Borough
Green, Dutta A.C.
1993. Botany for Degree Students. 5th edition. Ghazanfar, S.
1989. Savanna Plants: An Illustrated
Guide. Macmillan Publishers. 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. Kaufman
B. P., Carlson F.T., Dayananda P., Evans L.M.,
Fisher B.J., Parks C. and Wells R.J. 1989. Plants: Their Biology and
Importance. Harper and Row
Publishers, Reiner R.
1984. Antibiotic: An Introduction. New Horn Publishing Co., Ronald
M.A. 1995. Micro-organisms in our World. Mosby Yearbook, Inc., Sule I.O.
and Agbabiaka, T.O. 2008. Antibacterial effect of
some plant extracts on selected enterobacteriaceae.
Ethnobotanical Leaflets 12:1035-42. Table 1. Minimum inhibitory Concentration (MIC) of
the water extract on the clinical bacterial isolates.
- = No growth; + = growth Table 2. Minimum inhibitory
concentration (MIC) of the ethanolic extract on the
clinical bacterial isolates.
-, No growth; +,
Growth Table 3. Diameters of zones of
inhibition (mm) of the extracts at 10% w/v on the clinical bacterial
isolates.
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