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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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