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NEWPORT INTERNATIONAL JOURNAL OF BIOLOGICAL AND
APPLIED SCIENCES (NIJBAS)
Volume 5 Issue 1 2024

https://doi.org/10.59298/NIJBAS/2024/5.1.606711
Page | 60
Exploring the Antibacterial Properties of Vernonia
amygdalina (Bitter Leaf) Extract: A Potential Alternative
to Conventional Antibiotics
David Musyoki
School of Pharmacy, Kampala International University, Uganda

ABSTRACT
As part of the ongoing search for potent and resistance-free antibacterial medicinal plants, this study aimed to
evaluate the antibacterial properties of the plant extract of Vernonia amygdalina, commonly known as bitter leaf.
Standard procedures were used to provide a potential cheap alternative to conventional medication for treating
bacterial infections. The aqueous extract of V. amygdalina leaves was prepared and subjected to phytochemical
screening, which revealed the presence of tannins, phlobatannins, saponins, terpenoids, cardiac glycosides and
alkaloids. The antibacterial activity of the extract was tested against the gram-positive bacterium Staphylococcus
aureus and the gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli using the agar well diffusion
method. The extract showed moderate antibacterial activity, exhibiting 10 mm and 8 mm zones of inhibition
against S. aureus and P. aeruginosa respectively at a concentration of 20 mg/ml. However, it displayed no activity
against E. coli. In comparison, the standard antibiotic gentamicin produced larger zones of inhibition of 33.5 mm,
27 mm, and 23.5 mm against the respective test organisms. The results suggest that V. amygdalina extract had
greater antibacterial activity on the gram-positive S. aureus than on the gram-negative microorganisms tested. The
presence of phytochemicals like tannins, saponins and alkaloids in the extract may contribute to its antibacterial
properties. Further research is warranted to fully elucidate the medicinal potential of V. amygdalina and isolate the
active compounds responsible for the observed antimicrobial effects. Overall, the findings provide a scientific basis
for the traditional use of this plant in treating bacterial infections.
Keywords: Vernonia amygdalina, Antibacterial activity, Phytochemicals, Antimicrobial resistance, Traditional
medicine

INTRODUCTION
The search for novel, effective, and affordable antimicrobial agents has become a global priority as many infectious
disease-causing bacteria are developing resistance to commonly used synthetic antibiotics [1]. This growing
antimicrobial resistance poses a significant public health concern, especially in developing countries where access
to modern medicine is limited. In response to this challenge, researchers are increasingly turning their attention to
exploring natural plant-based sources for alternative antimicrobial therapies. Traditional herbal medicine has a
long history of use in many Asian, Latin American, and African countries, where up to 80% of the population
relies on plant-derived remedies as their primary healthcare [2, 3, 4]. These traditional practices are founded on
empirical observations passed down through generations, suggesting that certain medicinal plants possess
pharmacological properties beneficial for treating various ailments, including infectious diseases. One such plant
that has garnered attention for its potential antimicrobial applications is Vernonia amygdalina, commonly known
as bitter leaf. V. amygdalina is a tropical shrub indigenous to sub-Saharan Africa that has been used extensively in
traditional medicine [5]. Its leaves, roots, and other plant parts are reported to possess a variety of therapeutic
effects, such as antihelminthic, antimalarial, laxative, and fertility-enhancing properties [6, 7, 8]. Furthermore, the
leaves are commonly consumed as a vegetable in many African cuisines after undergoing processing to reduce their
characteristically bitter taste [9]. Interestingly, observations of wild chimpanzees in Tanzania chewing V.
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amygdalina leaves to extract the bitter juice and subsequently returning to normal activity have lent support to
the plant's medicinal value [10]. Traditional healers in various parts of Africa also commonly use V. amygdalina
leaf extracts to treat bacterial infections and chronic skin ulcers, even in cases where antibiotic treatments have
failed [11 - 15].
Previous studies have investigated the antibacterial properties of V. amygdalina extracts against various bacterial
strains. Akinpelu [16] found the plant extract to be effective against Bacillus subtilis, Klebsiella pneumoniae,
Pseudomonas aeruginosa, Proteus vulgaris, Shigella dysenteriae, and Staphylococcus aureus, but inactive against
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Escherichia coli and Serratia marcescens. Similarly, Ashebir and Ashenafi [17] reported that a 7% V. amygdalina
extract inhibited the growth of B. cereus, S. aureus, and Shigella flexneri, but had no effect on E. coli. Vernonia
amygdalina has also been shown to contain various secondary metabolites, such as tannins, saponins, alkaloids, and
terpenoids, which have been associated with antimicrobial properties in other medicinal plants [6, 16, 17].
However, the exact mechanisms by which V. amygdalina exerts its antimicrobial effects, as well as the specific
bioactive compounds responsible, remain to be fully elucidated. Further investigation is needed to provide a more
comprehensive understanding of the antibacterial potential of this plant and its potential applications in
traditional and modern medicine. The present study aims to build upon the existing evidence by evaluating the
antibacterial activity of aqueous extracts of V. amygdalina leaves against a panel of clinically relevant bacterial
pathogens, including S. aureus, P. aeruginosa, and E. coli. The findings of this research could contribute to the
development of plant-derived antimicrobial agents and provide a scientific basis for the traditional medicinal use
of V. amygdalina.

METHODOLOGY
Study Site
This was an experimental study carried out at Kampala InternationalUniversity-Western Campus Pharmacy
laboratory.
Equipment and Materials
Petri dishes, aqueous leaf extract, electronic weighing balance, Distilled water, Beakers, Measuring cylinder, Pen,
Water bath, Refrigerator, Nutrient agar, McConkey agar, Spatula, Sterile cork borer, Incubator, Hot air oven.
Plant Material Identification
Vernonia amygdalina plant was identified by a botanist (Professor DominicByarugaba) at Kampala International
University-Western campus and avoucher sample was prepared as herbarium.
Preparation of the Extract (aqueous extraction)
Leaves of Vernonia amygdalina was collected around Ishaka. The leaves werethen air dried for 2 weeks, crushed,
and then blended. 12g of the leaves wereweighed and used in extraction. Extraction was done by dissolving the
groundleaves in 100ml of hot water boiled for thirty minutes in a conical flask andthen was soaked for 24 hours. It
was then filtered using filter paper and dried in a water bath to obtain the extract. The extract was weighed to
obtain the weight/weight.
Phytochemical Screening
Tests for the detection of different secondary metabolites were carried out usingaqueous extracts of Vernonia
amygdalinaaccording to standard procedures as described by [18, 19, 20].
i. Flavonoids
To 5mls of the dilute ammonia solution, 0.2g of Vernonia amygdalina aqueousextract was added followed by the
addition of 2ml concentrated sulphuric acid. Observation of a yellow solution that turns colourless was indicative
of flavonoids.
ii. Tannins
0.2g of the extract was dissolved in 5mls of distilled water and heated in a waterbath for fifteen minutes then
filtered. Three drops of 0.1 %ferric chloride wereadded to the filtrate and observed for the presence of a brownish-
green or blue-blackcoloration indicative of tannins.
iii. Saponins
0.2g of the extract was dissolved in 5 ml of distilled water and then heated toa boil. The formation of a layer of
foam indicated the presence of saponins.
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iv. Alkaloids (Mayer's test)
0. 5g of the extract was stirred in 1 % aqueous hydrochloric acid in a steambath for five minutes. Two to
three drops of Mayer's reagent were added to theside of the test tube. Turbidity or a white precipitate
with this reagent wastaken as evidence for the presence of alkaloids.
v. Phlobotannins
0.2g of the extract was dissolved in 5 ml of distilled water and filtered. Thefiltrate was boiled with 2 ml of 2% of
aqueous hydrochloric acid solution. Observation of a red precipitate was considered a positive test for
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Phlobotannins.
vi. Steroids
0.2g of the extract was mixed with 2 ml of concentrated sulphuric acid andthereafter2 ml of acetic anhydride was
added to the mixture color changed fromviolet to blue or green indicatingthe presence of steroids.
vii. Cardiac glycosides
A drop of ferric chloride solution was added to 2ml of glacial acetic acid. Thissolution was used to treat 0.5g of
the extract. The mixture was then underlaidwith 1 ml of concentrated sulphuric acid. A brown ring at the
interphase wasindicative of a deoxy-sugar characteristic of cardenolidesA. A violet ring mayalso appear below the
brown ring while in the acetic acid layer, a greenish ringmay form gradually throughout the thin layer indicating
the presence ofcardiac glycosides.
viii. Reducing sugars
0.2g of the extract was shaken with 5ml of distilled water and filtered. Thefiltrate was then boiled with 3 drops
of Fehling's solution A&B for two minutes. An orange solution indicated the presence of a reducing sugar.
Plant Extract Disc Preparation
The plant extract disc was prepared from a labline filter by punching with a corkborer of 6mm diameter and the
disc was autoclaved at 121 degrees Celsius for 15minutes. 0.2g of the extract was then diluted with l0mls of
distilled water togive a concentration of 20mg/ ml and this was added to the discs. Thisconcentration was chosen
as it correlated with the concentration of thestandard antibiotic (gentamycin) that was to be used. The plant
extract discwas then dried in an oven and stored in a refrigerator until required for use.
Culturing of the Test Microorganism
The isolates of the microorganism were obtained from the MicrobiologyLaboratory of Kampala International
University-Western campus. The testorganisms used were Staphylococcus aureus, Pseudomonas
aeruginosaandEscherichia coli. Staphylococcus aureus and Pseudomonas aeruginosamicroorganisms were cultured on
Nutrient Agar plates by dissolving 2.8g ofNutrient Agar in l00mls of water while Escherichia coli was cultured
inMcConkey agar. The media was autoclaved at 121 degrees Celsius for 15 mins.9mls of this media was poured in
plates and left to gel. The microorganismswere later sub-cultured to produce young cultures which were used in
theexperiment.
Determination of Antibacterial Activity
i. Agar well diffusion method
This was carried out according to the method described by Opara and Anasa (1993). The growth media Mueller-
Hinton Agar (MHA) was prepared bydiluting 9g of Mueller-Hinton agar in 250 ml of distilled water and
sterilizing byautoclaving. MHA was allowed to cool to 50 degrees Celsius and 5 ml of themolten agar was then
added to the petri dishes. Three wells of about 6.0mmdiameter were aseptically made on each agar plate using a
sterilecork borer. The cultured microorganisms were later inoculated on the Mueller-Hinton Agarby spreading the
microorganisms uniformly across the Petri dishes. Fixedvolumes (20µl) of the plant extract were then introduced
into the wells. Controlwells containing gentamycin were used as a positive control because it is abroad-spectrum
antibiotic and was set up at the center and distilled water wasthen added to the third well as a negative control.
The plates were incubated at5 degrees for 1 hour to ensure the extract and the controls diffused evenly inthe agar
and then were incubated at 37 degrees Celsius for 24 hrs. The relativesusceptibility of the microorganisms to the
plant extract was determined bymeasuring the zones of inhibition and the experiment was done two times
forstaphylococcus aureus and Escherichia coli plates and once for Pseudomonas aureginosa plates and the mean values
were calculated and recorded.
Statistical Analysis of Data
Test for significance in the zone of inhibition was done by determining themean of the zone of inhibition
produced by the bacteria to know theeffectiveness of each plant extract and the susceptibility of the test organism.
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Limitations of the Study
The research results were not as expected due to the failure to isolate and sterilizethe active principles due to a
lack of equipment.
Time Limit
The research study took six months beginning from March 2011 to August 2011.
Sources of Chemicals and Reagents
Chemicals and reagents for phytochemical screening and other reagents wereprovided by the School of Pharmacy,
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Kampala International University-WesternCampus.
RESULTS
Extraction
After extraction of 12g of leaf extract in l00mls of distilled water, the yield was3.3g of extract. The percentage
yield obtained was 27.5% w /w i.e. 3.3/ 12 x 100.
Table 1: Phytochemical test

(+) refers to the presence of the phytochemical and (-) refers to the absence of the phytochemical.
Table 2: Antimicrobial activity of extracts of Vernonia amygdalina determined by agar well diffusion
method on specific media for each testmicroorganism

Aqueous extract:(-) = no activity, P. aeruginosa= Pseudomonas aureginosa, Staph. aureus=

Staphylococcus aureus, Esch. coli = Escherichia coli
DISCUSSION
Medicinal plants are used by a large proportion of developing nations [20-30]. The reason for this may be a true
improvement in disease conditions after herbal treatment [31-40]. In these countries, the search for new drugs is
centered upon theinvestigation of medicinal plants [41-50]. The present research has tested the crude extracts of
V. amygdalina onbacterial strains which comprised of Staphylococcus aureus, Pseudomonasaeruginosa, and Escherichia
coli. The therapeutic effect of some species of plants is determined by their constituents [31-45]. These
constituents increase the body's resistance to disease, retardor delay the processes of natural aging or facilitate the
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adaptation of theorganism to certain conditions [22]. Phytochemical screening analysis revealed the presence of
tannins, phlobatannins, saponins, terpenoids, glycosides, and alkaloids in the plantextract. Elsewhere it is reported
that tannins, alkaloids, saponins, flavonoids, and glycosides could be associated with the antimicrobial activities of
someplants [23, 24]. The antibacterial activities of these plants may reside in these active principles as noted by
[25]. What has not been resolved is the separation of thespecific bioactive components against specific organisms;
and this has been noted to affect the quality and safety of herbal medicines [26]. The aqueous extract also showed
no presence of phytochemicals such asflavonoids, steroids, and reducing sugars. Vernonia amygdalina extracts had Page | 64
inhibition on Staphylococcus aureus (grampositive) and Pseudomonas aeruginosa (gram negative) but had no
inhibition onEscherichia coli (gram negative). This is similar to the observation of the plants'ability to inhibit E.
coli [17]. The aqueous extract of Vernonia amygdalinahad slightantibacterial properties at a percentage of 30%
activity on Staphylococcusaureus (10mm zone of inhibition) when compared to the standard antibioticactivity of
gentamycin at 100% (33.5mm) and 29% activity on Pseudomonasaeruginosa (8mm zone of inhibition) when
compared to the standard antibioticactivity of gentamycin at 100% (27mm). The extract showed no activity
onEscherichia coli and could not be compared with the standard antibiotic. The extract may be used as an adjuvant
to the standard treatment. However, such an addition of therapy needs to be explored whether associated with any
drug interactions. The difference in antimicrobial properties of a plant extract is attributable tothe age of the
plant used, freshness of plant materials, physical factors (temperature, light water), contamination by field
microbes, adulteration andsubstitution of plants, incorrect preparation and dosage [28, 29]. Vernonia amygdalina
showed a greater inhibitory effect on the gram-positive (S. aureus) than on the gram-negative strains (P. aureus)
[17, 30, 31]. The standard antibiotic used which was gentamycin showed zones of inhibition on E. coli, P.
aeruginosa, and S. aureus. Gentamycin had the greatest effect on Staphylococcus aureus (33.5mm zone of inhibition),
then on Pseudomonasaeruginosa (27mm zone of inhibition), and lastly on Escherichia coli (23.5mmzone of
inhibition). Gentamycin showed a stronger retardation effect on the gram-positive bacterial strains than on the
gram-negative ones. Distilled water which was used as a negative control showed no effects oneither of the
bacterial strains.
CONCLUSION
This study revealed that the Vernonia amygdalina aqueous extract at 20 mg/ mlhad shown slighter antibacterial
action when compared with the vehicle-treatedgroup. However, when compared to standard antibiotics, it had only
producedone-fourth of activity. Moreover, it is active against only two organisms such asPseudomonas aeruginosa,
and Staphylococcus aureus but not on Escherichiacoli. Hence this plant extract may be used only as an adjuvant to the
standardtreatment, provided it does not produce any drug interactions.
RECOMMENDATIONS
Ethanol and other extractions of the plant extract should be carried out to ascertain the presence of other
phytochemicals and determine their antibacterial activity. It is essential to isolate the specific component
responsible for the antibacterial activity of the extract. Improving the quality of results will require upgrading
the equipment used in isolating the active principles. Furthermore, to comprehensively assess the antibacterial
efficacy of the extract, conducting minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) tests is recommended. Fractionation of the extract should also be pursued to identify which
phytochemical component exhibits the strongest antibacterial activity.
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CITE AS: David Musyoki (2024). Exploring the Antibacterial Properties of

Vernonia amygdalina (Bitter Leaf) Extract: A Potential Alternative to
Conventional Antibiotics. NEWPORT INTERNATIONAL JOURNAL OF
BIOLOGICAL AND APPLIED SCIENCES, 5(1): 60-67.
https://doi.org/10.59298/NIJBAS/2024/5.1.606711