Sky Journal of Microbiology Research Vol. 3(4), pp.

041 - 045, August, 2015

Available online http://www.skyjournals.org/SJMR
ISSN 2315-876X 2015 Sky Journals

Full Length Research Paper

Antibacterial activity of bitter leaf (Vernonia
amygdalina) soup on Staphylococcus aureus and
Escherichia coli)
Lovet T. Kigigha1* and Ebubechukwu Onyema2
Department of Biological Sciences, Niger Delta University Wilberforce Island, P. M. B., 71, Yenagoa, Bayelsa State,
Nigeria.
Accepted 25 July, 2015

The study was aimed at assessing the effect of cooking Egusi-soup on the antibacterial activity of bitter leaf
(Vernonia amygdalina). Extracts of the leaves of bitter-leaf plant were made using Ethanol, from Bitter-leaf soup
and using hot water. The antibacterial activity of the extracts was tested using two pathogenic hospital isolates
viz. Staphylococcus aureus and Escherichia coli. The antibiotic, Ampiclox (a broad- spectrum antibiotic in 1 mg
-1
ml concentration) was used as control. The test showed that all the three forms of extracts were inhibitory to
the two isolates in which the hot water extract indicated the highest inhibitory zone on E. coli and also with S.
aureus (P < 0.001). This was followed by the inhibitory effect of bitter leaf soup extract which was significantly
higher than the inhibitory effect of Ampiclox on E. coli (representing the enteric bacteria) (P = 0.025).
Interestingly, the effect of bitter leaf soup extract was also higher than the effect of Ampiclox on S. aureus
(representing superficial etiologic agents) indicating a probable positive effect on the health of probably
external and internal mucosa. The observed antibacterial effect of V. amygdalina on the selected bacterial
isolates appear to justify the traditional use of bitter leaf as a choice vegetable for African soup recipes and for
the cure of a number of human diseases (such as the treatment of gastroenteritis, fever and wound infections
etc).

Key words: Vernonia amygdalina, bitter leaf, antibiotic, Enterobacteriaceace, Escherichia coli, Staphylococcus aureus.

INTRODUCTION

Plants extracts are continuously being sort for as the roots of V. amygdalina have been used for gingivitis
effective and cheaper alternative sources of medication and toothache due to its proven antimicrobial activity.
all over the world especially in the developing countries. This study was designed to investigate the effect of
Vernonia amygdalina commonly called bitter leaf cooking Egusi soup (a major conduit for using bitter leaf
(because of its bitter taste) is consumed either as a in Nigeria and in Africa generally) on the antibacterial
vegetable for cooking African soups or the aqueous activity of the plant. This is because bitter leaf soup when
extracts could be drank as tonics for the treatment of consumed gives a great soothing, appetizing and
various illnesses (Imaga and Bamigbetan, 2003). The unusually satisfying after-effect.
bitterness is suspected to be due to factors such as the
presence of alkaloids, saponins, tannins and glycosides
which have been shown by various authors to be present LITERATURE REVIEW
in bitter leaf (Butler and Bailey, 1973; Ologunde et al.,
1992 and Bonsi et al., 1995). In the wild, chimpanzees Global use of medicinal plants
have been observed to ingest the leaves Vernonia plants
when suffering from parasitic infections. According to According to World Health Organization (WHO, 2007),
Huffman et al. (1993), medicinal plants are plants in which one or more of its
parts contain substances that can be used for therapeutic
purposes or which are precursors for the synthesis of
*Corresponding author. E-mail: [email protected]. useful drugs. The local use of natural plants for primary
42 Sky. J. Microbiol. Res.

health remedies is a common practice in Asia, Latin V. amygdalina. Fred et al. (2009), researched on the
American and Africa (Bibitha et al., 2002). Though many nutritional and antimicrobial properties of V. amygdalina
plants are consumed as food without an in-depth leaves. In the same year, Ibrahim et al. (2009) carried out
knowledge of their exact chemical composition and the assessment of the antibacterial activity of V.
contribution to health, their utilization through several amygdalina and Occimum gratissimum leaves on
generations appear to justify their use (Ghani et al., selected food borne pathogens. The aqueous and
1989). Use of plant extracts or their active principle may ethanol extracts of these plant leaves were tested against
serve as source of new drugs or sources of intermediate Escherichia coli, Staphylococcus aureus, Bacillus cereus,
compounds for synthesizing analog drugs (Akerele, Shigella dysentriae and Salmonella typhimurium in which
1993). the plants showed antibacterial activity on the entire test
isolates. Imaga and Bamigbetan (2013) carried out an in
vivo biochemical assessment of aqueous extracts of V.
Bitter leaf (Vernonia amygdalina) amygdalina (Bitter leaf). Indication was that there was an
improved functionality of the antioxidant system of the
Vernonia amygdalina, a member of the Asteraceae test rats due to the probable effect of the phytochemicals
family, is a small ever green shrub that grows in tropical and antioxidants in the plant extract. It was inferred that
Africa. It is a shrub of 1 - 3 m in height with petiole leaf of aqueous extract of V. amygdalina could be consumed as
about 6 mm in diameter and elliptical in shape (Igile et al., food or as an herbal medicine without appreciable toxicity
1995). The plant is mostly found in West Africa where the to body organs and tissues.
most used part is its leaves (called bitter leaf). The leaves The present study was designed to investigate the
are dark green in colour with a characteristic odour and effect of cooking Egusi soup (a major conduit for using
when chewed has a bitter taste but delicious in meals bitter leaf in Nigeria and Africa generally) on the
due to its pleasant nostalgic bitterness when it interacts antibacterial activity of the plant. This is because bitter
with protenous ingredients (such as fresh or dry fish) in leaf soup when consumed gives a great soothing,
the soup. Local names by which the plant is called in appetizing and unusually satisfying after-effect; thus it
Nigeria include: Kiriologbo (in Ijaw); Onugbu (in Igbo), would be of medicinal interest to ascertain the extent of
Ewuro (in Yoruba) and Shiwaka (in Hausa). depreciation or otherwise of the known antibacterial
The leaves are used as soup condiment and as activity of the plant when consumed in Egusi soup.
vegetable after crushing and washing off using water to
remove some of the bitterness (Mayhew and Penny,
1998). Other African soups, in which bitter leaf is used MATERIALS AND METHODS
apart from Egusi soup, include: Ogbono and Okra soups.
In many parts of west Africa, the leaves could also be
used for washing slime off fish and snail before cooking Preparation of plant extracts
while roots and twigs could be used as chewing-stick.
All parts of the plant are pharmacologically suspected Samples of fresh plant of V. amygdalina leaves were
to be useful. Both the roots and leaves are used in the bought at Amassoma market in Bayelsa state of Nigeria.
treatment of fever, hiccups, kidney disease and stomach The plant was used to prepare three different extracts: i)
discomfort, among others (Gill et al., 1992; Homoiona the leaf sample was rinsed three times with distilled and
and Saffaf, 1994). The plant is claimed to also exhibit deionized water. 100 g of the leaf (in three batches) were
anti-helmitic and anti-malaria properties (Abosi and pounded using sterile mortar and pestle. The first batch
Raserika, 2003) as well as anti-tumourgenic properties was soaked in 100ml of ethanol which was stoppered
(Izevbigie et al., 2004). Locally it is used in treating with cotton wool covered with aluminum foil and left for
stomach ache (for immediate relief). The expressed 24 h. in the refrigerator. This was then filtered using
extract is used in treating skin infection such as Whatman No.1 filter paper and the extract stored in the
ringworm, itching, rashes and eczema. It is also claimed refrigerator for use. ii) The second batch of 100 g of
to cure diabetes, loss of memory, pneumonia and pounded bitter leaf was filtered using 100 ml of boiled
arthritis. Studies by Oboh and Masodje (2009) indicated water and stoppered with cotton wool covered with
that V. amygdalina fresh leaf had moisture content of aluminum foil and left for 24 h in the refrigerator. The
830%; dry matter of 1702%; protein 130% and ash extract was then filtered using what man No.1 filter paper
content 050%). Its mineral content (per gram) is and stored in the refrigerator for use. iii) The third batch
Phosphorus 6155 g; Selenium 82 g; Iron 471 g and of 100 g pounded bitter leaf was used for preparing Egusi
Zinc 113 g. soup. Bitter leaf soup preparation was started with an
initial 800 ml of water. Ingredients used alongside with V.
Previous research works on Vernonia amygdalina amygdalina include; fresh pepper, salt, soup thickener,
dry fish, meat, crayfish, palm oil, onion, magi. After
Indication is that various studies have been carried out on cooking, the final volume of the soup was 100 ml.
 Kigigha and Onyema 43

Antimicrobial Activity of Bitterleaf

60 E.coli S.aureus

50
Zone of Inhibition mm 2

40

30

20

10

0
Et Ex Hot W BLS AMPI
Treatment of Bitterleaf
Figure 1. Effect of Bitter leaf on test organisms (S. aureus and E. coli) .Et Ex = Ethanol
Extract; Hot W = Hot water Extract; BLS= Bitter leaf Soup Extract
AMPI = Ampiclox Extract; (P = < 0001); Data = (Mean = SE); n = 3.

This was strained with sterilized and then filtered using prepared from Whatman No. 1 filter paper using cock
Whatman No.1 filter paper and the extract stored in a borer. The solution containing the extracts (0.1 ml each)
refrigerator for use.. were impregnated on the disc in triplicates using sterile
pipette and air- dried.
Media and apparatus
Antibacterial assessment of extracts
All media were prepared according to manufacturers
specifications while all glassware were washed with Using flamed-sterilized and cooled pair of forceps the
detergent and rinsed with deionized and distil water three discs which had been impregnated with the bitter-leaf
times and were then autoclaved for 15 min. extracts including the one percent Ampiclox control were
(in triplicates) placed on separate Nutrient agar plates
Test bacterial isolates which had been confluent inoculated with the test E. coli
and S. aureus isolates. The discs were well spaced in the
Clinical isolates of S. aureus and E. coli were obtained agar plates to avoid overlapping of the zones of inhibition.
respectively from Gloryland Clinic, Yenagoa, Bayelsa The plates were inoculated at 37C for 72 h to observe
state and the Department of Pharmaceutical Microbiology the zones of inhibition.
Laboratory, College of Health Sciences, Niger Delta
University, Bayelsa state. They were characterized with
respect to their morphological, biochemical and antibiotic RESULTS
susceptibility.
The result as shown in Figure 1, indicated that all the
Preparation of antibacterial discs from leaf extracts three extracts were inhibitory to the two bacterial test
isolates viz. E. coli and S. aureus (which represent the
The disk-diffusion technique was used to test the enteric forms and the superficial etiologic agents
antibacterial activity of the different extracts using 10 mm respectively). From Figure 1, the hot water extract
discs (Kigigha and Atuzie, 2010). Antibiotic discs were indicated the highest inhibitory zone on E. coli and also
44 Sky. J. Microbiol. Res.

Table 1. Morphological and biochemical characteristics of the test bacterial isolates.

Test and Assessment Staphylococcus aureus Escherichia coli

Colony Morphology Cocci Rod
Gram stain + ve - ve
Growth in MSA + ve NT
Coagulase test (tube/slide) + NT
Catalase test + NT
Sugar Utilization
Lactose NT +
Mannitol NT +
Glucose NT +
Sucrose NT +
EMB NT +
CLED NT +
Key: NT: = Not Tested; +ve: = Positive reaction; -ve: = Negative reaction.

Table 2. Antibiotics sensitivity of the test bacterial isolates.

Escherichia coli Staphylococcus aureus

Antibiotics Sensitivity Antibiotics Sensitivity
Nitrofurantion (N) ++ Ofloxecin (OF) +++
Ciprofloxacin (CIP) +++ Erythromycin (E) +++
Tetracycline (TE) R Ciprofloxacin (CIP) +++
Amoxicillin (AX) R Clindamycin (CD) R
Ofloxacin (OF) ++ Gentamycin (GN) +++
Chloramphenicol (C) + Cephaplexin (CX) R
Cefuroxine (CF) R Ominoxazola (CE) R
Ampicillin (AM) R Ampicillin/ CLOXAC ++
Gentamicin (GN) R Floxapen (FX) +
Norfloxacin (NB) R Augumentin (AU) +++
Key: + = Slightly positive; ++ = moderately positive; +++ = highly positive; R =
Resistant.

on S. aureus compared to the Ampiclox control (P < using SigmaStat statistical package. All pair wise multiple
0.001). This was followed by the inhibitory effect of bitter comparison procedures at level of Comparison P < 0.050,
leaf soup extract on E. coli, which was significantly higher (or Tukey Test) indicated as follows:
than the inhibitory effect of Ampiclox on S. aureus (P <
0.001) and also higher than the inhibitory effect of Hot W Ec vs. AMPI Sa (P <0.001); Hot W Ec vs. AMPI Ec
Ampiclox on E. coli (P = 0.025). (P<0.001);
In Table 1, the test isolates were characterized Hot W Ec vs. BLS Sa (P <0.001); Hot W Ec vs. Et Ex Ec
accordingly. The antibiotic resistance pattern in Table 2, (P = 0.002);
indicated that E. coli was resistant to six out of the ten Hot W Ec vs. Et Ex Sa (P = 0.004); Hot W Ec vs. Hot W
antibiotics tested viz. Tetracycline, Cefuroxime, Sa (P = 0.036);
Amoxicillin, Ampicillin, Gentamicin and Norfloxacin. BLS Ec vs. AMPI Sa (P = <0.001); BLS Ec vs. AMPI Ec
While the Staphylococcus isolate was resistant to three (P = 0.025);
out of the ten antibiotics tested viz. Clindamycin, Hot W Sa vs. AMPI Sa (P = <0.001); Hot W Sa vs. AMPI
Cephlexin and Ominoxazole. Ec (P = 0.036);
Et Ex Sa vs. AMPI Sa (P = <0.001); Et Ex Ec vs. AMPI
Sa (P = <0.001).
Statistical analysis

The differences in the mean values among the treatment Discussion

groups were greater than would be expected by chance;
there was statistically significant difference (P < 0.001) The antibiotic resistance pattern indicated that the test
 Kigigha and Onyema 45

isolates were resistant to a number of antibiotics; Huffman MA, Gotoh, S, Izutsu D, Koshimizu K, Kalunde MS (1993).
Further observations
invariably, they were viewed to have originated from
on the use of the medicinal plant, Vernonia amygdalina (Del) by a wild
some pathogenic forms. The inhibition on both E. coli and chimpanzee,its possible effect on parasite load, and its
S. aureus, indicated that extraction of bitter-leaf with hot phytochemistry. Afr. Study Monogr. 14(4): p227-240.
water, was significantly higher than that obtained from Ibrahim TA, Ajala L, Adetuyi, FO, Jude-Ojei B (2009): Assessment of
the antibacterial activity of Vernonia amygdalina and Ocimum
bitter-leaf soup extraction. This appears to indicate a
gratissimum leaves on selected foodborne pathogens. EJEAFChe
probable positive effect on the health status of enteric 8(11):1212-1218.
etiologic agents and on the superficial internal and Igile GO, Oleszek W, Jurzysta M, Burda S, Fafunso M, Fasanmade AA
external mucosa. Of the known 17 species of the genus (1994). Flavonoids from Veronia amugdalina and their antioxidant
activities. J. Agric. Food Chem., 42:24452448.
Vernonia all have been shown to exhibit therapeutic
Izevbigie EB, Bryant JL, Walker A (2004). A novel natural inhibitor of
characteristics viz. as blood purifier, uterus toner and also extra cellular signal related kinases and human breast cancer cell
to prevent atherosclerosis (Erasto et al., 2007; Nwanjo, growth. Experimental Biology Medicine (Maywood). Exp. Biol. Med.
2005). Many herbalists and naturopathic practitioners 228:293298
Kigigha LT, Atuzie MN (2012). Assessment of traditional medicinal
recommend aqueous extracts for their patients as
application of Alchornea cordifolia. Afr. J. Biotechnol., 11(8). 2083
treatment for anaemia, nausea, diabetes, loss of appetite, 2086.
dysentery and other gastrointestinal track ailments. V. Kupchan SM, Hemingway RJ, Karim A, Werner D (1969). Tumor
amygdalina extracts have also been reported to help inhibitor XLVII Vernodalin and vernomygdin, two new cytotoxic
sesquiterpene lactones from
suppress, delay, or kill cancerous cells (Kupchan et al.,
Vernonia amygdalina. J. Org. Chem., 34(12): 3908.
1969). Latha SP, Kannabiran K (2006). Antimicrobial activity and
The present study showed that the cooking of Egusi phytochemicals of Solanum trinobatum linn. Afr. J. Biotechnol.,. 5
soup (a major conduit for using bitter leaf in Nigeria and (23): 2402-2404.
Madunagu BE, Ebana RUB, Udo SM, Ndifon LT (2001). Antimicrobial
Africa generally) decreased the antibacterial activity of effects of Ixora divaricata and Citrus aurantifolia on some pathogens
the plant probably due to the presence of different and drug resistant Neisseria
protenous ingredients such as dried fish, crayfish etc. But gonorrhoeae. Nig. J. of Bot., 14:63-69.
then it was yet to be ascertained if the presence of the Mayhew S, Penny A (1988). Macmillian Tropical and Subtropical Foods.
Macmillian publishers, London.
same protenous ingredients may not be responsible for Nwanjo H (2005). Efficacy of aqueous leaf extract of Vernonia
detoxifying bitter leaf extract and thus giving the soothing amygdalina on plasma lipoprotein and oxidative status in diabetic rat
and pleasant feeling of satisfaction that accompanies models. J. of Physiol. Sci., 20:39-42.
bitter-leaf Egusi-soup consumption. The study also Oboh FOJ, Masodje HI (2009). Nutritional and antimicrobial properties
of Veronia amygdalina leaves. Int. J. Biomed. and Health Sci., 5: 51 -
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57.
vegetable and as a medicinal plant. Ologunde MO, Ayorinde FO, Shepard RK, Afolabi OA, Oke OL (1992).
Sterols of seed oils of Vernonia galanesis, Amaranthus cruentus,
Amaranthus caudatus, Amaranthus hybridus and Amaranthus
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