Ibrahim and Ogaji., Nig. Journ. Pharm. Sci., September 2012, Vol. 11 No.2, P.

41–49

Nigerian Journal of Pharmaceutical Sciences

Vol. 11, No.2, September, 2012, ISSN: 0189-823X
All Rights Reserved

CRUDE FLAVONOIDS FROM VERNONIA KOTSCHYANA POSSESS

ANTIMICROBIAL ACTIVITY

* Ibrahim, G. and Ogaji, Y. N.

Department of Pharmacognosy and Drug Development, Faculty of Pharmaceutical Sciences,

Ahmadu Bello University, Zaria- Nigeria

Author for correspondence: garzaky@yahoo.com Phone: +2348035960302

ABSTRACT
Vernonia kotschyana Sch. Bip. (Asteraceae) is an annual herb or a shrub that grows up to 2 meters high and it is
widely distributed in both the southern and northern part of Nigeria. It is used ethno-medically in the management
of stomach ache, gonorrhea, gingivitis, arthritis and tuberculosis. Phytochemical screening was carried out on
leaves ethanol extract and crude flavonoids of the plant by using standard phytochemical methods. Antimicrobial
studies were also carried out on the crude flavonoids of the plant by using agar well diffusion and tube dilution
methods. The leaves ethanol extract of the plant was found to contain saponins, carbohydrates, tannins, steroids and
flavonoids. The crude flavonoids of the plant within concentrations (100 - 6.25 mg/ml) used had significantly
(p<0.05, 0.01) inhibited the growth of Staphylococcus aureus, Escherichia coli, Aspergillus niger and Candida
albicans and had respective minimum inhibitory concentrations (MIC) of 12.5, 6.25, 25.0 and 25.0 mg/ml. Zones
of growth inhibition produced by the crude flavonoids of the plant were less than those produced by Ofloxacin (20
µg/ml) and Fluconazole (25 µg/ml) that were respectively used as standards for bacterial and fungal strains. The
crude flavonoids of the plant could serve as a potential source of antimicrobial agent based on the results of the
present study.

Keywords: Phytochemical, Antimicrobial, Crude flavonoids, Vernonia kotschyana, Asteraceae

INTRODUCTION The Genus Vernonia has about 1000

Vernonia kotschyana Sch. Bip is of the species. Phytochemical constituents
family Asteraceae. It is an annual herb or a associated with this genus include
shrub that grows up to 2 meters high and it alkaloids, carbohydrate, tannins,
is widely distributed in both the southern saponins, flavonoids and volatile oils
and northern parts of Nigeria. It is used (Nwanjo and Ojoku, 2005).
ethno-medicinally in the management of Biological activities of some members
some ailments such as stomach ache, of the genus Vernonia include:
gonorrhea, gingivitis, arthritis and antioxidant, analgesic,
tuberculosis (Burkill et al., 1997). antihypertensive, antidiabetic, anti-
diarrhea and anti-pyretic effects (David et

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Ibrahim and Ogaji., Nig. Journ. Pharm. Sci., September 2012, Vol. 11 No.2, P. 41–49
al., 1993; Anson et al., 2001; Nwanjo and
Ojoku, 2005).
The roots are prepared in Northern
Nigeria into a bitter medicine which is used
as a digestive, appetizer and as remedy for
stomachache while in Southern Nigeria, the
roots are used as chew-sticks whereas in
Mali they used the plant for the treatment of
gastrointestinal disorders and wound healing
(Adjanohoun et al., 1985).
The ethanol extract of V. kotschyana
was reported to be active against a wide
variety of bacterial species including
Salmonella species and Staphylococcus
aureus. Its extract was found to contain
alkaloids (Deeni and Hussain, 1994). The
gastro protective properties of V. kotschyana
root showed effect against chemical-induced
injury in-vivo. The aqueous extract is active
on experimental ulcers induced by restraint
cold, ethanol and indomethacin (Germano et
al., 1996; Sanogo et al., 1996).
Phytochemical constituents are responsible
for the identities and varied biological
activities observed in a great number of
medicinal plants (Trease and Evans, 1983).
In general, susceptibility is accepted by
most workers to mean that an organism will
be killed or inhibited in precise quantity in-
vitro by the concentration of the drug, which
is easily attainable in the blood (WHO,
2002).
For the increasing cost and wide range
of side effects associated with a number of
synthetic antimicrobial drugs, there is a need
for a search of an alternative source of
42
antimicrobial agents from natural products.
This study was aimed at evaluating the
antimicrobial potentials of the crude
flavonoids of this plant.
MATERIALS AND METHODS
Plant Collection, Identification and
Preparation
The plant was collected at Zaria in July,
2011 and was identified in the Herbarium
section of Biological sciences, Ahmadu Bello
University Zaria with voucher specimen
number 6915. The plant was dried and
powdered using pestle and mortar. The
resultant powder was subsequently referred
to as the powdered plant material.
Phytochemical Studies
Extraction of Plant Material
Powdered leaves of V. kotschyana (350
g) was weighed and defatted with petroleum-
ether (60-80) in a percolator after which it
was filtered. The marc was subjected to
maceration with ethanol (96% v/v) for 24
hours and filtered. The filtrate obtained was
then evaporated to dryness on a water bath
(75oC) which yielded crude leaves ethanol
extract (18.5g) in accordance with the
methods outlined by Brain and Turner
(1975) and Trease and Evans (1983).
Fractionation of the Extract
The crude leaves ethanol extract was
then subjected to fractionation to obtain the
crude flavonoids in accordance with Woo et
al.(1980) as indicated in Figure 1.
Ibrahim and Ogaji., Nig. Journ. Pharm. Sci., September 2012, Vol. 11 No.2, P. 41–49

350g of Vernonia kotschyana Powdered Leaves

Pet – ether extraction

Marc
Pet- ether extract Ethanol Extraction
(Maceration)

Ethanol Extract Marc

Diluted with H2O and

Partitioned with Diethyl
Ether (1:5)

Ether Fraction Aq. Fraction

Partitioned with n-
Butanol
Saturated with Water

n- Butanol Fractn. Aq. Fraction

Partitioned with 1% KOH

n- Butanol (Saponins) KOH Fraction

Acidified with dil. HCl and

Partitioned with n- Butanol
Saturated with Water

HCl Fraction n- Butanol (Flavonoids)

(Flavonoids)

Figure 1: Schematic Chart for Fractionation of Flavonoids from Vernonia

kotschyana Powdered Leaves (Woo et al., 1980).

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Ibrahim and Ogaji., Nig. Journ. Pharm. Sci., September 2012, Vol. 11 No.2, P. 41–49
Chemical Tests for the Crude
Flavonoids
Different qualitative chemical tests
namely: Ferric chloride (FeCl 3), Lead
acetate, Sodium hydroxide and Shinoda
were used to detect the flavonoids fraction
as outlined by Brain and Turner (1975).
Antimicrobial Studies
Preparation of Culture Media
The media used were prepared according to
manufacturer’s instructions and these
comprised: Nutrient agar (Antec. Diagnostic
Products, U. K), Saboraud Dextrose Agar
(Oxoid Ltd, Basingstoke, Hampshire;
England) and Nutrient Broth (Fluka
Chemical Co., Spain).
Collection of Microbial Cultures
Cultures of Staphylococcus aureus,
Escherichia coli (bacteria) and Aspergillus
niger, Candida albicans (fungi) strains were
collected from Pharmaceutical Microbiology
Laboratory, Ahmadu Bello University,
Zaria. The bacterial cultures were collected
on the prepared nutrient agar slants while
the fungal cultures were collected on the
prepared Saboraud dextrose agar slants
(Hugo and Russell, 1998).
Standardization of the Cultures
Using the prepared normal saline, the gram
positive bacterium: Staph. aureus was
serially diluted to a concentration of 1: 1000
while the gram negative bacterium: E. coli
was serially diluted to a concentration of 1:
5000 (Bertina and Wentworth, 1987).
Fungal species A. niger and C. albicans
were maintained on Saboraud dextrose agar,
incubated at 25oC for 24 hrs. The fungal
growth was harvested, washed and the
inoculum was standardized to 1x 106
cells/ml (Sanaa et al., 2007).
44
Preparation of the Crude Flavonoids
Dried (in desiccators) crude flavonoids (1.0
g) of the plant was weighed and dissolved in
10 ml of sterile distilled water to make up a
stock solution of 100 mg/ml. Thereafter, 0.1
ml each of 100, 50, 25, 12.5 and 6.25 mg/ml
concentrations were prepared and stored in
the refrigerator until needed for use.
Susceptibility Test
The agar cup diffusion method was used for
this test. Sterile nutrient agar plates were
flooded with appropriately diluted organism,
the excess was aseptically drained and the
surface was allowed to dry at room
temperature. Wells were bored into the agar
plates using a 4 mm sterile cork borer and
0.1 ml each of different concentrations of
the crude flavonoids (100 – 6.25 mg/ml) was
introduced into each well. The standards
used were Ofloxacin and Fluconazole at
20 and 25 µg/ml respectively. The plates
were allowed a pre-diffusion time of 1 hr at
room temperature and then incubated at
37°C and 30°C for 24hr for bacterial and
fungal strains used respectively. Thereafter,
zone of inhibition for each well was read to
the nearest millimeter and recorded (Sahm
and Washington, 1990).
Minimum Inhibitory Concentration
(MIC)
Minimum inhibitory concentration (MIC)
was determined using the tube dilution
method of Sahm and Washington (1990).
Crude flavonoids solution (1.0 ml) at
concentrations used (100 – 0.195 mg/ml)
was added to 1.0 ml of nutrient broth and
1.0 ml was transferred from the first tube to
the next up to the ninth tube. Then, 0.1 ml of
24 hr. culture of standardized test organisms
was inoculated into each test tube and mixed
thoroughly. The tube with the lowest
dilution with no detectable growth was
considered as the minimum inhibitory
concentration.
Ibrahim and Ogaji., Nig. Journ. Pharm. Sci., September 2012, Vol. 11 No.2, P. 41–49

Statistical Analysis
Antimicrobial activity of the crude
flavonoids of the plant was expressed as
zones of inhibitions for the different
concentrations used as compared with those
produced by the standards (Ofloxacin and
Fluconazole) by using student t-test at p <
0.05 and 0.01 (Michael and Robert, 2003).
RESULTS
Phytochemical Studies
Phytochemical constituents identified in the
crude leaves ethanol extract of the plant
include: carbohydrate, tannins, saponins,
steroids and flavonoids (Table 1). Crude
flavonoids fraction (Figure 1) of the plant
gave positive coloured chemical reactions
with the various detecting reagents used for
determining their identities as shown in
Table 2.

Table 1: Some Phytochemical Constituents Identified in Ethanol Leaf Extract of

Vernonia kotschyana
Constituent Test Observation Inference
Carbohydrate Molisch’s Purple to violate colour +
Fehling’s Brick red ppt. +

Phenolic FeCl3 Greenish black colour +

compounds

Saponins Frothing Froth formed +

Steroids Liebermann-Burchard Brownish ring +

Salkwoski's Reddish brown +

Flavonoids Shinoda's Orange colour +

NaOH Yellow colour +
FeCl3 Greenish colour +

Alkaloids Dragendorff’s Orange-red ppt. -

Wagner’s Brown ppt. -
Mayer’s White ppt. -
Key: + = present; - = absent

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Ibrahim and Ogaji., Nig. Journ. Pharm. Sci., September 2012, Vol. 11 No.2, P. 41–49

Table 2: Chemical Tests for the Crude Flavonoids Fractionated from the Leaves Ethanol
Extract of Vernonia kotshcyana
Constituent Test Observation Inference
Flavonoids FeCl3 Greenish precipitates +
Lead Acetate Yellow precipitates +

NaOH Yellow colour +

Shinoda Yellow colour +
Key: + = present

Susceptibility Test Ofloxacin (45 and 48 mm) against Staph.

Significant (p < 0.05 and 0.01) zones of
growth inhibition (mm) against Staph.
aureus of 25, 22, 18 and 16 mm and E.
coli of 28, 24, 20 and 15 mm were
respectively observed at 100, 50, 25 and
12.5 mg/ml concentrations in both cases
whereas those against A. niger were 18
and 16 mm at 100 and 50 mg/ml and for
C. albicans were 20, 18 and 15 mm at
100, 50 and 25 mg/ml as produced by the
crude flavonoids of the plant. These were
compared with those produced by
aureus and E. coli at 20 µg/ml and
Fluconazole (50 and 55 mm) against A.
niger and C. albicans at 25 µg/ml
respectively (Table 3).
Minimum Inhibitory Concentration
(MIC)
The minimum inhibitory concentrations of
the crude flavonoids of the plant against
Staph. aureus, E. coli, A. niger and C.
albicans were respectively found to be 12.5,
6.25 and 25.0, 25.0 mg/ml (Table 4).

Table 3: Zones of Inhibition (mm) Produced by Crude Flavonoids from Vernonia

kotschyana against the Test Organisms

Organism Zones of Inhibition (mm) at Various Crude Flavonoids Standard

Concentrations (mg/ml) Oflox. Fluco.
100 50 25 12.5 6.25 20 25 (µg/ml)
Staph. aureus 25** 22** 18* 16* 12 45

E. coli 28** 24** 20* 15* 12 48

A. niger 18* 16* 14 10 NZ 50
C. albicans 20* 18* 15* 12 NZ 55
Keys: NZ = No zone of Inhibition; Significant at *p<0.05, **p<0.01 using student t-test
E. coli = Escherichia coli, Staph. aureus = Staphylococcus aureus, A. niger = Aspergillus niger, C. albican
= Candida albican,

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Ibrahim and Ogaji., Nig. Journ. Pharm. Sci., September 2012, Vol. 11 No.2, P. 41–49

Table 4: Minimum Inhibitory Concentration (MIC) of Crude Flavonoids from

Vernonia kotschyana against the Test Organisms

Orgs. Concentrations of Crude Flavonoids of Vernonia kotschyana (mg/ml)

100 50 25 12.5 6.25 3.12 1.56 0.78 0.390 0.195 MIC

55 5 2 1
SA - - - - + + + + + + 12.5

EC - - - - - + + + + + 6.25

AN - - - + + + + + + + 25.0

CA - - - + + + + + + + 25.0

Keys: SA = Staphylococcus aureus, EC= Escherichia coli, AN = Aspergillus niger,

CA = Candida albican; + = No inhibition of growth; - = Inhibition of growth,

DISCUSSION crude flavonoids (Table 3). This suggests that

The different phytochemical constituents
identified in the leaves ethanol extract of V.
kotschyana are considered as significantly
important biologically active compounds of
plant origin. This may be important in the
discovery of new therapeutic agents,
especially at this time when the scientific
community is preoccupied with searching for
alternative treatment to combat the increasing
threat of drug resistant micro-organisms. The
phytochemical constituents: tannins,
saponins, and flavonoids are secondary
metabolites that are responsible for various
pharmacological activities including
antimicrobials of many medicinal plants
(Trease and Evans, 1997).
The antimicrobial studies had shown that
both bacterial and fungal strains tested were
susceptible to the crude flavonoids of the
plant as significant (p<0.05 and 0.01) zones
of growth inhibition were produced at various
concentrations (100 – 6.25 mg/ml) used.
However, it was found that the bacterial were
more susceptible than the fungal strains as
shown by their respective zones of growth
inhibitions (15 – 28 and 15 – 20 mm) to the
the plant might have contained phytochemical
with potential to serve as a broad spectrum
antimicrobial. Hugo and Russell (1998) states
that the spectrum of activity of antimicrobial
agent depends on how wide the agent is able
to inhibits the active proliferation of micro-
organisms.
The antimicrobial activity displayed by
the crude flavonoids as zones of growth
inhibition against the tested microbes was
concentration dependent. This reveals that the
antimicrobial activity of the crude flavonoids
as there was proportionate decrease in zones
of growth inhibition with decrease in its
concentrations (Table 3). The mode of action
of flavonoids can either be by formation of
complexes with the extra-cellular and soluble
proteins of the bacteria or its cell wall thereby
destroying it (Cowan and Steel, 1993).
The minimum inhibitory concentration
(which is the lowest concentration that
inhibits the growth of micro-organisms) of the
crude flavonoids observed (6.25 – 25.0
mg/ml) against Staph. aureus, E. coli, A.
niger and C. albicans (Table 4) indicated that
it is more effective against E. coli (6.25

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Ibrahim and Ogaji., Nig. Journ. Pharm. Sci., September 2012, Vol. 11 No.2, P. 41–49
mg/ml) than S. aureus (12.5 mg/ml) and even
A. niger and C. albicans (25.0 mg/ml).
Successful antimicrobial therapy of an
infection ultimately depends on the
concentration of antibiotic at the site of
infection. This concentration must be
sufficient to inhibit growth of the offending
microorganism. If host defenses are intact
and active, a minimum inhibitory effect,
such as that provided by bacteriostatic
agents may be sufficient. On the other hand,
if host defenses are impaired, antibiotic-
mediated killing may be required to
eradicate the infection (Goodman and
Gillman, 2006).
Some bacteria show inherent or innate
resistance to certain antibiotics (e.g.
Pseudomonas aeruginosa is always resistant
to benzyl penicillin). Other bacteria have
acquired resistance as a result of genetic
change (e.g. some strains of Streptococcus
pneumonia are now resistant to penicillin).
Significant increases in bacterial resistance
have been seen recently, and some strains of
Staphylococci, Streptococci and Gram
negative rods have been identified that are
resistant to all currently available
antibacterial agents (Steven et al., 1999).
CONCLUSION
Crude flavonoids of Vernonia kotschyana
was found to have significant and
concentration dependent antimicrobial
activity as such it could therefore, serve as
a potential source of antimicrobial agent of
medicinal plant origin.
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