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ABSTRACT
Introduction
Vernonia amygdalina commonly known tropics and other parts of Africa
as bitter leaf (English), Oriwo(Edo), particularly Nigeria, Cameroon, and
Ewuro (Yoruba), Shuwaka (hausa), and Zimbabwe. The leaves are dark green
Olubu (igbo), is a tropical shrub that coloured with a characteristics odour and a
grows up to 3 meters high in the African bitter taste. It is reputed to have several
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All the clinical isolates were sub cultured match the turbidity (opacity) standard
on Nutrient and MacConkey agar medium prepared as described by Monica (1984).
respectively for purity and was maintained
on nutrient agar slant at 4c in the
refrigerator until when required for use. Determination of minimum inhibitory
concentration (MIC) of the extract on
Sample preparation and extraction the test organisms
10g of dried powder of the plant material The initial concentration of the plant
was added to 100ml of sterile distilled extract (100g/ml) was diluted using double
water or 70%w/v ethanol in order to obtain fold serial dilution by transferring 5ml of
water or ethanol extracts (100mg/ml). The the sterile plant extract (stock solution)
extraction was done at room temperature into 5ml of sterile nutrient broth to obtain
for 24 hours for the water extract and 50mg/ml concentration. The above process
72hours for the ethanol extract (Newton et was repeated several times to obtain other
al., 2002). Muslin cloth was then used to dilutions: 25mg/ml, 12.5mg/ml,
filter the plant residue and the filtrate thus 6.25mg/ml and finally 3.125mg/ml
obtained was further purified by filtration (Ibekwe et al., 2001). Having obtained the
through whatman no.1 filter paper (Atata different concentrations of the extract,
et al.,2003).The stock solution of the each concentration was inoculated with
extract was then sterilized by filtration 0.1ml of the standardized bacterial cell
through Millipore membrane filter of suspension and incubated at 37c for
0.45m pore size (Ronald, 1995). The 24hours. The growth of the inoculum in
sterile extract obtained was then stored in broth is indicated by turbidity or
sterile capped and refrigerated at 4c until cloudiness of the broth and the lowest
when required for use. concentration of the extract which
inhibited the growth of the test organism
Sterility proofing of the extract were taken as the Minimum Inhibitory
Concentration (MIC). Negative control
The extract was then tested for sterility was set up as follows: nutrient broth only;
after Millipore filtration by introducing nutrient broth and sterile plant extract, and
2ml of the sterile extract into 10ml of finally positive control containing nutrient
sterile nutrient broth. This was incubated broth and a test organism.
at 37c for 24hours. A sterile extract was
indicated by absence of turbidity or Determination of zone of inhibition
clearness of the broth after the incubation
period (Ronald, 1995). Fifteen millimeter (15ml) of sterile
nutrient agar was poured into each sterile
Standardization of the bacterial cell Petri dish of equal size and allowed to
suspension solidify. The surface of this sterile nutrient
agar plate was streaked with the pure
Five colonies of each test organism were culture of the standardized bacterial cell
picked into sterile test-tube containing suspension. A cork borer (8mm in
sterile nutrient broth and incubated at 37c diameter) was sterilized by flaming and
for 24hours. The turbidity produced by was used to create ditch at the Centre of
this organism was adjusted and used to the plate. The hole so created was then
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filled with the plant extract. The plate was sample was weighed into a conical flask in
allowed to stand for one hour for pre- which 10ml of sterile distilled water was
diffusion of the extract (Esimone et al., added and boiled for 5minutes. The
1998) and incubated at 37c for 24hours. mixture was filtered and 2.5ml of the
At the end of the incubation period, the filtrate was added to 10ml of sterile
diameter of the zone of inhibition was distilled water in a test tube. The test tube
measured in millimeter using meter rule was stopped and then shaken vigorously
(Hugo and Russel, 1996). for about 30seconds. It was allowed to
stand for half an hour. Honey comb froth
Phytochemical Analysis of the leaves indicates the presence of Saponins.
extracts
Test for Phenol
The Phytochemical screening of the leaves
extracts viz; Flavonoids, Alkaloids, 25ml of extract was added to 2ml of ferric
Saponins, Phenol, Glycosides, Volatile chloride solution, a deep bluish green
Oil, Tannins, Steroids and Terpenoids, solution formed indicates the presence of
Anthraquinone, Cardenolide was analyzed phenol.
using the method described by Harbourne
(1983), Trease and Evan (1989), and Test for Glycosides
Sofowora (1993), respectively.
25ml of 1ml Sulphuric acid was added to
Test for Flavonoids 5ml of the extract in a test tube and boiled
for 15minutes, cool and neutralized with
Sodium hydroxide method was used for 10% sodium hydroxide, and then 5ml of
the test. 5g of the sample was weighed and fehling solution A and B was added. A
detanned completely with acetone. The brick red precipitate of reducing sugars
residue was extracted in warm water after indicates the presence of Glycosides.
evaporating the acetone on a water bath.
The mixture was filtered and the filtrate Test for Tannins
was used for the test. 5ml of 10% sodium
hydroxide was added to an equal volume 3g of the sample was boiled in 50ml
of the detanned water extract. A yellow distilled water for 30minutes on a hot
solution indicates the presence of plate. The mixture was filtered and a
Flavonoids. portion of the filtrate was diluted with
sterile water in a ratio of 1:4 and 3drops of
Test for Alkaloids 10% ferric chloride solution was added. A
blue or green colour indicates the presence
2mls of the extract was measured in a test of tannins.
tube to which picric acid solution was
added. The formation of orange Test for volatile oils
colouration indicates the presence of
alkaloids. 2ml of extract solution was shaking with
0.1M sodium hydroxide and a small
Test for Saponins quantity of 0.1M hydrochloric acid. A
white precipitate was formed with volatile.
Froth test for saponins was used. 1g of the
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Herbs that have tannins as their Neumann et al (2004) also confirmed the
component are stringent in nature and are antiviral properties of steroids, Quilan et al
used for treating intestinal disorder such as (2000) worked on steroidal extracts from
diarrhea and dysentery (Dharmananda, some medicinal plant which exhibited
2003). This observation therefore supports antibacterial activities on some bacterial
the use of Vernonia amygdalina in herbal isolates.
cure remedies.
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Alkaloids which are one of the largest Okoli, A.I 2008. The bioactive and
groups of phytochemical in plant have phytochemical properties of
amazing effect on human and have led to Garnicia Kola Hackel seed extract on
the development of powerful pain killer some pathogens Afr.J. of
medicine (Kam and Liew, 2002). One of Biotechnol. 721:3934-3938.
the most common biological properties of Atata, R.F., Sani, A. and Ajewole, S.M
alkaloids is their toxicity against cells of 2003. Effect of Stem Bark extracts of
foreign organisms. Enantia chloranta on some
Clinical Isolates. Nig. Society for
In conclusion, this study showed that Experimental Biology. 15 2: 84-
Vernonia amygdalina (bitter leaves) 92.
extract exerted significant (P<0.05) Ademola, I.O., Eloff, J.N 2011.
antimicrobial activities against the tested Antihelminthic activity of acetone
clinical isolates and might be source of extract and fractions of
active antimicrobial agents for the Vernonia amygdalina against
development of drugs caused by these Haemonchies contortus eggs and
pathogens. However, the presence of these larvae. Trop. Arum. Health prod.
phytochemical compounds in Vernonia 432: 521-527.
amygdalina (bitter leaves) leaves extract Adesanoye, O.A., Farombi, E.O 2010.
identified in this study could be attributed Hepatoprotective effect of Vernonia
to the antifungal and antibacterial amygdalina astereacea in rats
activities observed. treated with carbontetrachloride, EXP,
Toxicol, pathol 62:197-206
Recommendations
[pubmed].
Blanco, J.G., Gul, R.R., Bocco, J.L.,
1. Based on this study, it is recommended
Meragelman, T.L., Gentiraimondi, S.,
that people should develop the habit of
Flury, A 2001. Aromatase
eating bitter leaf because it has immense
inhibition by an 11, 13-
medicinal values in the treatment of
Dihydroderivative of a
various human pathogens.
sesquiterpene lactone .J.
2. It is also recommended that the plant
pharmacol, Expt., therap; 297:1099-
should be used for the development of
1105.
drugs for the curing of pathogens tested in
Cimanga, R.K., Tona, L., Mesia, K.,
this study.
Musuamba, C.T., De Bruyne,
3. It is recommended that further study
T.,Apers, S., Hernan, N., Miert
should be conducted on the mechanisms of
,V.S., Pieters, L., Totte, J., Viletink,
action of other parts of the plant viz; root,
A.J 2004. In vitro
stem, bark, e.t.c.
antiplasmodia activity of extract
and fraction of seven medicinal plant
References used in the democratic republic
of Congo .J. Ethnopharmacol 93:27-
Abort, A.O., Raserika, B.H 2003. In vivo 32 [pubmed].
antimalarial activity of Vernonia Dharmananda, S. 2003. Allnuts and the
amygdalina. British Journal of uses of tannins in Chinese medicine in
Biomedical Science; 60:89-91 proceedings of institutes for
[pubmed]. traditional medicine port. 3938 land
Adegboye, M.F., Akinpelu, D.A and
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