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Abstract
The leaves of Crotalaria lachnosema
were freshly collected, dried under-shade and ground into powder. The ethanolic
extract of the sample was obtained by cold extraction and was fractionated with
solvent of varied polarity. The fractions were analyzed for their
phytochemicals and screened antimicrobial against Staphylococcus aureus,
Salmonella typhi and Escherichia coli. The phytochemicals were
distributed among the test fractions. Tannins were found to be present in all
the fractions and methanol fraction contains all the other tested
phytochemicals except alkaloids and cardiac glucosides. The activities of the
fractions were found to be more pronounced against E. coli than against the
other test organisms.
Keywords: Phytochemical Screening; Antimicrobial; Crotalaria
lachnosema; Staphylococcus aureus; Salmonella aureus; Salmonella typhi;
Escherichia coli; Klebsiella pneumoniae
Introduction
For many centuries, man explores and
utilizes the natural endowment offered by both the species of flora and fauna
to provide the basic necessity of life such as clothing, shelter, food and
indeed health care. Medicinal plants are the richest and commonest natural
resource used in traditional medicine. Of the 250, 000 higher plant species on
earth, more than 80,000 are medicinal [1]. Although plants had been priced for
their medicine, flavoring effect and aromatic qualities for centuries, but the
synthetic products of the modern age had for some time surpassed their
importance. However, the blind dependence on synthetics is over and people are
returning to the naturals with hope of safety and security [1]. The development
of drug resistance in human pathogens against commonly used antibiotics has
necessitated a search for new antimicrobial substances from other sources including
plants [2]. Many reports have attested the efficacy of herbs against
microorganisms, as a result, plant is one of the bedrocks of modern medicine to
attain new principles [3]. The therapeutic properties of plants may not be
unconnected to the variety of chemical substances biosynthesized by the plants
as “secondary metabolites’’ that bring about definite physiological action in
the human body. The most important of these bioactive constituents of plants
are alkaloids, tannins, flavonoids, saponins and etc. [4]. Presently many
governments and major health institutions including the World Health
Organization [5] have recognized, pharmacologically validated and improved many
traditional herbal medicines and eventually integrated them in formal health
care system [1]. Thus, in light of the evidence of rapid global spread of
resistant clinical isolates, the need to find new antimicrobial agent is of
paramount importance. However, the past record of rapid, widespread emergence
of resistance to newly introduced antimicrobial agents, indicates that even new
families of antimicrobial agents will have a short life expectancy [6]. For
this reason, researchers are increasingly turning their attention to herbal
products, looking for new leads to develop better drugs against MDR microbe
strains [7].
Crotalaria lachnosema belongs to the
family Fabaceace (Leguminoseae),
sub-family Papilionoideae. It is a woody plant with a height of about 2
cm high. The plant is known as ‘Fara birana’ in Hausa, ‘komp’ in Yoruba, ‘Ake
dinwo’ in Ibo and Birjibei in Fulani [8]. The genus Crotalaria is widespread in
the tropics and subtropical region and has about 550 species [9]. C. lachnosema
was found to be important in the treatment of scabies. The whole plant grounded
and mixed with water are fed to animals to treat liver disease [8]. The
presence of resins and balsams might support the use of the plant as emollient
as well as for treatment of sore throat, rheumatism, wounds and burns. Since
some basalms and resins has antiseptic properties [3]. Few species of
Crotalaria have been assessed against some pests. For example, under greenhouse
condition, C. retusa and C. juncea have been found to be
resistant to attack by the nematode, Pratyylenchus zeae and also that C.
retusa has shown a higher degree of resistance to attack by the nematode, Rotylenchus
rnifirmis Linford and Olivera. It was also reported that, the non-polar
extract of C. retusa contain some active ingredients for controlling
flea beetle a pest on okro plant. So, could be useful in pest management [10].
Materials and Methods
Sampling
and Sampling Sites
The leaves of Crotalaria lachnosema
were freshly collected on 4th July 2011 at an uncultivated land in
Damanko village about 9km west of Zaria main town, Zaria Local Government, Kaduna
State. The plants were identified and authenticated by Mallam Umar Shehu Galla
of the Herbarium unit, biological science, Ahmadu Bello Univesity, Zaria. The
leaves of the plant were dried under-shade for seven days and ground into
powder using clean pestle and mortar.
Extraction
and Fractionation of Plant Materials
Cold extraction (Percolation) was
adopted in this research, this is part of the appropriate measure to preserve
constituents that may potentially be active and retain their original identities
in the course of preparing the extract [11]. 200g of the powdered plant sample
was weighed and sucked in1000cm3 of ethanol for 14 days. The crude extract was
prepared by decantation, filtration and concentration of the filtrate using
Rota vapor machine (RVO) at 400C and finally by drying the concentrated crude
ethanol extract. Fractions of various degrees of polarities were obtained from
ethanol extract by macerating the ethanol extract with different solvents in
sequence starting with solvent of least polarity to the one of highest polarity
[12]. For the fractionation, 30cm3 of n-hexane was poured into the beaker that
contained the dried and gummy ethanol extract and stirred for 5minutes and the
liquid portion was then drained into another cleaned and empty beaker. This
process was repeated until a clear solution was obtained at the end. The entire
procedure was repeated with other solvents in the series; chloroform, ethyl
acetate and methanol. Four fractions were thus obtained from the exercise and
were labeled as followed: n-hexane fraction, chloroform fraction, ethyl acetate
fraction and methanol fraction.
Phytochemical
Screening of Plant Sample
The phytochemical analyses of the
fractions were conducted by subjecting the fractions to different standard confirmatory
tests. This is to determine the presence of certain phytochemical classes.
Test for Alkaloids: Each fraction (0.5g) was stirred with 5ml of 1 percent
aqeous hydrochloric acid on a steam bath; 1ml of the filtrate was treated with
a few drops of Mayer’s reagent and a second 1ml portion was treated similarly
with Dragendoff’s reagent. Turbidity or precipitation with either of these
reagents was taken as evidence for the presence of alkaloids in the extract
being evaluated [13].
Test for Saponins: Each fraction (0.5g) was shaken with water in a test tube.
Frothing which persists on warning confirmed the presence of saponins [14].
Test for Tannins: Each fraction (0.5g) was stirred with 10ml of water. This
was filtered, and ferric chloride reagent was added to the filtrate, a
blue-black precipitate indicated the presence of tannins [15].
Test for Flavonoids: A portion of each fraction was heated with 10ml of
ethylacetate over a steam bath for 3mins. The mixture was filtered and 4ml of
the filtrate was shaken with 1ml of dilute ammonia solution. A yellow
colouration indicated the presence of flavonoid.
Test for Reducing Sugar: 1ml of each fraction was taken in five separate test tubes.
These were diluted with 2ml of distilled water followed by addition of
Fehling’s solution (A+B) and the mixtures were warmed. Brick red precipitate at
the bottom of the test tube indicated the presence of reducing sugar [16].
Test for Cardiac Glycosides: 2ml of each fraction was placed in a sterile test tube.
This was followed by adding 3ml of 3.5% iron III chloride (FeCI3), then 3ml
ethanoic acid. This gave a green precipitate and a dark colored solution
respectively. Finally, concentrated H2SO4 was carefully poured down the side of
the test tub e which resulted in the formation of brownish red layer, at the
interface. This confirms the presence of cardiac glycosides.
Antimicrobial
Activity Test
Agar disc diffusion technique was
adopted for the sensitivity test as described by [17].
Preparation of Test Fractions’
Concentration: Discs of about 6mm diameter were
punched from Whatman’s No 1 filter paper using a paper puncher. Batches of 10
of the paper discs were transferred into vial bottles and sterilized in an oven
at 1400C for 60 minutes. Stock solutions of 100mg/ml of the fractions were
prepared by dissolving 200mg of each fraction in 2ml of DMSO (Dimethyl
sulphoxide). By means of 1ml sterile syringe, 0.1ml, 0.2ml, 0.5ml and 1.0ml
were transferred into labeled vial bottles preoccupied with 10 paper discs from
a stock solution of each fraction and the solution were subsequently diluted
with 0.9ml, 0.8ml, 0.5mland 0.0ml (i.e. without dilution) of DMSO that
correspondingly resulted to 1mg/disc, 2mg/disc, 5mg/disc and 10mg/disc
concentration. The prepared concentrations of the test fractions in the labeled
bottles were kept in refrigerator until required for use.
Preparation of Inoculum from the
Test Micro-Organisms: Staphylococcus aureus,
Salmonella typhi, Escherichia coli and Klebsiella pneumoniae that
were sourced from Microbiology unit of Aminu Kano Teaching Hospital (AKTH)
Kano, were the microorganisms used for the research. The identities of the
microorganisms were confirmed by standard biochemical test [18]. The test
organism was cultured and maintained in a nutrient agar slant at 40C. The
organism was then inoculated into nutrient broth and incubated overnight at
370C for 24 hrs. They were then diluted with normal saline until they give
concentration of bacterial cells equivalent to 0.5 McFarland standard of Barium
sulphate solution (1% v/v) [19].
Antibacterial
Susceptibility Test (Bio Assay)
A suspension of nutrient agar (28g
in 1000ml of distilled water) was prepared and autoclaved at 1210C for 15mins
according to the manufacturers’ instruction. It was then carefully poured into
sterile petri-dishes and allowed to solidify. The standardized inoculums of the
bacteria were swabbed on the surface of the solid nutrient agar plates by means
of sterile wire loop for the confluent growth of the bacteria. Four paper discs
of 10mg/disc, 5mg/disc, 2mg/ disc, 1mg/disc concentrations were taken from the
prepared test fraction solutions and were carefully and aseptically placed on
the inoculated surface of the nutrient agar and a positive control disc
(Tetracycline 1mg/disc) was placed at the centre of the plate. The plates were
incubated inverted at 370C for 18 hours. The diameters of clear areas
surrounding the discs where growths of the organisms were impeded (Zone of
inhibition) were measured in millimeter and recorded. The assay was repeated
two more times. The mean and the standard deviation (±SD) for the triplicate
values were then calculated.
Results and Discussion
Tables 1-3 Mean of the triplicates ±
S.D (standard deviation). A total ethanolic extract of 16.05g was produced from
the 200g powdered plant sample. The highest percentage mass (63.05%) of the
total mass macerated was methanol fraction and the least percentage mass
(0.56g) was the pet. ether fraction. The result of phytochemical analysis
revealed the availability of some secondary metabolites in the fractions of the
plant sample. The presence of these secondary metabolite’s accounts for the
activities of the plants. This complied with several reports by researchers
that plants contain bioactive substances. Tannins were detected in all the
fractions of the plant sample and tannins were reported to have various
physiological effects like anti-irritant, anti secretolytic, antiphlogistic,
antimicrobial and antiparasitic effect. Phytotherapeutically, tannins
containing plants are used to treat non-specific diarrhea, inflammations of
mouth and throat and slightly injured skins [20-22]. While cardiac glucosides
which are used as lexative and carthatic drugs were confirmed in chloroform and
ethyl acetate fractions. Alkaloids that were present in n-hexane and chloroform
fractions act as antimalarial and anti-amoebic agents [22]. The antimicrobial
sensitivity test result revealed a varied degree of activities exhibited by the
fractions of the plant against the test organisms. Although, the plant sample
exhibited low activities when compare to the control, the results show that
activity of the different fractions may increase further if the concentrations
of the fractions were to be increased. The result also showed that the
activities of the plant fractions were comparatively more pronounced against E.
coli than against S. aureus, S. typhi. and K. pneumoniae.
With the exception of chloroform fraction that demonstrated some activities
against S. aureus with zone of inhibition of 12mm at1000ug/disc all
other fractions were inactive against S. aureus. However, n-hexane and
ethyl acetate fractions exhibited low activities against S. typhi.
Conclusion
The activities of the fractions of the plant sample are more pronounced
against E. coli than against the other test organisms. E. coli
can cause diarrhea, urinary tract infections, respiratory illness, bloodstream
infections and other illness. So, the plant leaves can be used in the treatment
of the aforementioned illnesses. However, the relative low activities of the
plant sample fractions against S. typhi and K. pneumoniae
revealed its un-befitting nature as an antityphoid and anti-pnuemoniie drug.
Recommendation
The other parts of the plant should also be exploited. To harness its full
medicinal potential, the plant sample fractions should be tested against other
bacteria isolates and further research should be carried out to isolate and
characterize the active compounds in the plant.
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