ABSTRACT

This project is design to determine the phytochemical analysis and the anti-microbial effects
of vernonia amygdaline extracts on some selected pathogenic microorganism. Data will be
reported as mean + standard error of mean (SEM) and will be analyzed statistically using the
way ANOVA followed by Turkey-Kramer multiple comparison test and values o f p< 0.001
and 0.05 will be considered significant. And of the end of this research it is expected that the
extracts has inhibitory effect and selected micro organism at different concentration.

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INTRODUCTION

Vernonia amygdalina is a genus of about 1000 species of forbs and shrubs in the
family Asteraceae. Some species are known as ironweed. Some species are edible and
of economic value. They are known for having intense purple flowers. The genus is
named for the English Botanist William Vernon. There are numerous distinct
subgenera and subsections in this genus. This has led some botanists to divide this
large genus into several distinct genera (Harold, 2014).

Several species of Vernonia, including V. calvoana, V. amygdalina and V. colorata, are

eaten as leaf vegetables. Common names for these species include bitter leaf Onugbu
in the lgbo language, Ewuro and ndole. They are common in most West African and
Central African Countries. They are one of the most widely consumed leaf vegetables
of Nigeria, where the Onugbu soup is a local delicacy of the Igbo people, and of
Cameroon, where they are a key ingredient of Ndole. The leaves have a sweet and
bitter taste. They are sold fresh or dried and are a typical ingredient in Egusi soup
(Jucelia et al, 2013).

Vernonia amygdallna is well known as a medicinal plant with several uses attributed
to it, including management of diabetes, fever reduction and recently a non-
pharmaceutical solution to persistent fever, headache and joint pain associated with
AIDS (an infusion of the plant is taken as needed). These leaves are exported from
several African country and can be purchased in grocery stores aiming to serve
African clients. The roots of Vernonia amygdallna have been used for gingivitis and
toothache due to its proven antimicrobial activity (Austin, 2010).

Phytochemical are non - nutritive plant chemicals that have protective or disease
preventive properties. They are non essential nutrients meaning that they are not
required by the human body for sustaining life. It is well - known that plant produce
these chemicals to protect them but recent research demonstrates that they can also
protect humans against disease. There are more than thousand known phytochemicals.
Some of the well known phytochemicals are lycopene in tomatoes, isoflavones in
soybeans and flavonoids in fruits (Rosa et al, 2012).

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HISTORICAL BACKGROUND OF VERNONIA AMYGDALINA

Vernonia amygdalna is a multipurpose and rapid regenerating soft wooded shrub of 1

to 2Cm tall with petiolate leaves of around 6mm in diameter. This plant has been
named differently by different ethnics around the world. It is a species under the genus
Vernonia schreb (Family: compositae; Order: asterales; SIC: Asteridae; Classes:
Dicotyledons) which contains about 1000 species. More than 500 of these Vernonia
plants are distributed in Africa and Asia, approximately 300 in Mexico, Central and
South America and around 16 can be found in the United States (Jagun et al, 2015).

AIMS OF THE STUDY

This project is designed to determine the phytochemical analysis and antimicrobial

effects of extracts of on selected pathogenic microorganism.

OBJECTIVES OF THE STUDY

To determine the bioactive metabolite that vernonia amygdalina possesses by

phytochemical screening

To investigate which of the extracts (ethanolic or acetonic) that have high

antimicrobial activity on specific organisms

STATEMENT OF THE PROBLEM

Vernonia amygdalina is commonly used in traditional medicine for various purposes

due to its potency. There is need to identify the main chemical constituents responsible
for its potency in treatment of gastrointestinal problem, skin diseases such as rashes,
eczema, ring worms etc.

SCOPE OF THE STUDY

This research work is limited to phytochemical analysis and antimicrobial effects of

extracts of Vernonia amygdalina on selected pathogenic organisms.

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MATERIALS AND METHODS

MATERIALS

Test tube, Conical flask, Incubator, Autoclave, filter paper, Measuring cylinder,
Beakers, Water bath, Spatula, Cotton wool, Inoculating loop, Petri dishes, Funnel,
Cork borer and Round bottom flask

REAGENTS
Nutrient agar, Nutrient broth, Ethanol, Acetone, Distilled water, Sulphuric acid,
Chloroform, Fehlings A and B, Hydrochloric acid, Ferric chloride solution, Sodium
hydroxide, Potassium hydroxide, Dragendorff reagent, Wagner reagent and Mayers
reagent

PROCESSING OF THE PLANT LEAVES

The leaves will be processed according to the method of Atata et al (2011). The fresh
leaves will be collected by hand plucking from plant and cleaned of debris. The leaves
will be then air-dried at room temperature for 9days. The dried leaves will be blended
using a manual grinder. Powdered samples will be stored in tightly closed reagent
bottles for subsequent extraction and bioassay.

PREPARATION OF EXTRACTS

The method described by Akerele et al (2012) will be used to extract the bioactive
compounds from the powdered samples. The powdered samples will be extracted with
ethanol and acetone. About 20g portion of the leaf powder, two hundred milliliters
(200ml) each of ethanol and acetone will be added in separate conical flask, shaken
and covered with cotton wool and allowed to soak at room temperature for 24hours.
The extracts will be then filtered using filter paper and the two separate extracts
(ethanol and acetone) will be concentrated on water bath at 40C and stored in an
airtight bottle until required for used.

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STERILITY TEST OF THE EXTRACT

The ethanolic and acetonic extracts of the plants will be tested for sterility using the
method of Dalitha (2010). About 1mI of each extracts will be added into test tube
containing 5m1 of sterile nutrient broth. They will be then incubated for about 37C
for 24hours. The extracts will be clear after incubation indicating the absence of
contaminant which would have caused a turbid appearance in the tube.

TEST ORGANISMS

The test organisms will be clinical isolates of E. Coli (Escherichia coli),

Staphylococcus aureus, Kiebsielia pnuemonia and Pseudomonas aeruginosa. The
isolates will be obtained from General Hospital Nassarawa, Nassarawa State. The
organisms will be maintained on nutrient agar slant under refrigeration temperature.

PHYTOCHEMICAL ANALYSIS

Phytochemical qualitative analysis will be conducted on the ethanolic and acetonic

extracts of the leaves of Vernonia amygdalina to detect the presence or absence of
tannins, saponins, alkaloids, flavonoids and glycosides as described by Amadi et at
(2010).

TEST FOR TANNINS

About 3 drops of 5% ferric chloride solution will be added to 3m1 extract in the test
tube. A greenish - black coloration will emerge indicating the presence of tannins
(Trease et al, 2011).

TEST FOR GLYCOSIDES

To a small portion of the extract dilute sulphuric acid will be added and boiled in
water for l5minutes, cooled and 20% potassium hydroxide solution will be added and
divided into two (2) portions.

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(i). About 5ml of solution of Fehling A and B will be added to the first tube. The
formation of Brick- red precipitate will be formed indicate the presence of glycone
(sugar) moiety

(ii). About 3ml of ferric chloride will be added to test 2, the formation of blue
precipitate will be formed indicate the presence of phenolic nucleus (glycone moiety)
(Amadi et al, 2010).

TEST FOR ALKALOIDS

About 5ml of 2% hydrochloric acid will be used to dissolve little quantity of the
extract; the solution will be divided into 3 portions.

(i).To the first portion in the test tube, 2 drops of Dragendorffs reagent will be added.
Orange-brown precipitate will emerged indicating the presence of alkaloids.

(ii).To the second portion in the test tube, 2 drops of Wagner reagent. Brown
precipitate will be formed indicating the presence of alkaloids.

(iii).To the last portion in the test tube, 2 drops of Mayers reagent will be added.
Whitish precipitate will be formed indicating the presence of alkaloid (Harbone,
2010).

TEST FOR SAPONINS

About 2m1 of extract will be shaken vigorously with 5m1 of water and allowed to
stand for 45minutes. Persistent honeycomb will be formed which indicate the presence
of saponins. (Fafunso et al, 2011).

TEST FOR FLAVONOIDS

About 2m1 of 10% sodium hydroxide will be added to 3ml extract and 4 drops of
dilute hydrochloric acid will be added. Yellow color solution on addition of acid will
be formed which indicate the presence of flavonoids (Trease et al, 2011).

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ASSAY FOR ANTIMICROBIAL ACTIVITIES

About 2m1 of each of the inoculums will be transferred into the prepared nutrient agar
plates. The plates will be swirled properly and allowed to stay for some minutes. A
sterile cork borer (4mm in diameter) will be used to bore three wells on the inoculated
plates lettered A, B and C respectively. A 1.0ml concentration of ethanol extract will
be transferred into well A, while 0.5ml of the other concentrations will be transferred
into wells B and 0.25ml will be finally transferred to the well C. The same procedure
will be carried out for each organism for acetone extract. The plates will be incubated
at 37C for 24hours thereafter examined for zone of clearance around the wells, which
will be then measured, and the mean values recorded (Marshall, 2010).

DETERMINATION OF THE MINIMUM INHIBITORY CONCENTRATION

(M.L.C)

Determination of the minimum inhibitory concentration will be carried out using the
broth dilution method as described by Dalitha (2010). The MIC of the extracts will be
determined for the test organisms in triplicates at varying concentrations. A stock
solution of 10mI will be prepared for each extract separately. One milliliter (1ml) of
nutrient broth will be dispensed into test tubes and sterilized using an autoclave at
121C in 15minutes. The different extracts will be serially diluted from their stock
solutions to obtain varying concentrations. The concentrations will be: 1.0ml, 0.5ml,
0.25ml and 0.l ml of each test isolate will be inoculated into the various test tubes
containing varying concentrations and then incubated at 37C for 24hours. After
incubation the presence or absence of growth on each tube will be rated.

STATISTICAL ANALYSIS

Data will be reported as mean + standard error of the mean (SEM) and will be

analyzed statistically using One way ANOVA followed by Tukey- kramer multiple

comparison test and values of p< 0.001 and 0.05 will be considered significant.

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EXPECTED RESULT

The result is expected to show that both ethanolic and acetonic extracts contained alkaloids,
tannins, saponins and glycosides, while only flavonoids will be absent in both extracts. And
both of the extracts (ethanol and acetone) of the leaves had highest zones of inhibition on
Kiebsiella pnuemonea and Pseudomonas aeruginosa will be intermediate while Escherichia
coli and Staphylococcus aureus will be resistant to it. This resistance could be as a result of
the lower concentrations of the extracts used (0.2, 0.5 and 1.0mg/ml).

The Result of Phytochemical Analysis of Extract Vernonia amygdaline will be shown in the
table below:

BIOACTIVE ETHANOL EXTRACT ACETONE EXTRACT

COMPONENTS
Tannins + or - + or -

Saponins + or - + or -

Alkaloids + or - + or -

Flavonoids + or - + or -

Glycosides + or - + or -

KEY: (+) Presence, (-) Absence

The result of Minimum inhibitory concentration of the ethanolic and acetonic extracts will be
shown in the table below:

EXTRACTS CONCENTRATION E.coli Staphylococcus Pseudomona Kiebsielia

OF EXTRACTS aureus s aeruginosa Pnuemoni
(mg/ml) a
Vernonia Acetone 1.0 + or - + or - + or - + or -
amygdalina
0.5 + or - + or - + or - + or -
0.25 + or - + or - + or - + or -
Ethanol 1.0 + or - + or - + or - + or -
0.5 + or - + or - + or - + or -
0.25 + or - + or - + or - + or -

KEY: (+) Growth, (-) No Growth

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CONCLUSION

This research will show that the antimicrobial activity of Vernonia amygdalina using different
solvent extracts: ethanol and acetone on selected pathogens. However, local ethno medical
preparation and prescriptions of the plant sources should be scientifically evaluated and
disseminated properly. And the research work tend to suggests the need for more extensive
studies on these plants as sources of natural product for future use in the management of
multi-drug resistant pathogens such as Escherichia coli, Kiebsiella pnuemonea,
Staphylococcus aureus and Pseudomonas aeruginosa that cause wide range of infections.

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Amadi B.A, Agomuo, E.N and Ibegbulem, C.O (2010): Phytochemical Tests
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89-95

Atata, R.F. Sani, A and Ajewole, S.M. (2011): Effect of Stem Bark Extracts of
Enantia Chloranta on some Clinical Isolates. Nigeria Society for
Experimental Biology. 15(2): 84-92.

Austin, DF . (2000): Book Reviews. Econ. Bot, 54:234 245.

Fafunso, MA. And Morebise, O. (2011): Antimicrobial and Phytotoxic Activities

of Saponin Extracts from two Nigerian Edible Medicinal plants.
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Harold, R. (2014) : Generic and Subtribal Classification of American

Vernoniene (PDF) Smithsoman Contributions to Botany. 10:89-92.

Jucelia, B. and Santos, T. (2013): Vernonia Condensata Baker (Asteraceae): A

Promising Source of Antioxidants, Oxidative Medicine and Cellular
Longevity Article 69-80.

Marshell, K. (2010): Antimicrobial Susceptibility and Plasma Mediated

Streptomycin Resistance to Plesinemonia Shigelloid isolated from Blue
Crab. Journal of Applied Bacteriology 2:21-24.

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