Phytochemical and Antibacterial Studies of Root Extract of Cochlospermum
tinctorium A. Rich. (Cochlospermaceae)
Methanol extract of the root of Cochlospermum tinctorium
was evaluated for antibacterial activities using hole-in-plate bioassay
technique against Escherichia coli, Staphylococcus aureus,
Klebsiella pneumoniae, Corynbacterium ulcerans, Proteus
mirabilis and Shigella dysentriae using ciprofloxacin (10 Î¼g
mL-1) and gentamicin (10 μg mL-1) as reference
standards. The extract was active on all the test organisms at concentration
of 2000 μg mL-1. The activity of the extract against S.
dysentriae was found to be more potent with MIC 100 and MBC 500 μg
mL-1. Time kill studies showed that the antibacterial activities
were time dependent. Phytochemical screening revealed the presence of
alkaloids, flavonoids, tannins and cardiac glycosides. These phytochemicals
could be responsible for the antimicrobial activities exhibited by the
extract and hence justify the ethnomedicinal uses of C. tinctorium.
to cite this article:
M.B. Tijjani, I.A. Bello, A.B. Aliyu, T. Olurishe, S.M. Maidawa, J.D. Habila and E.O. Balogun, 2009. Phytochemical and Antibacterial Studies of Root Extract of Cochlospermum
tinctorium A. Rich. (Cochlospermaceae). Research Journal of Medicinal Plants, 3: 16-22.
For centuries, medicinal plants have been used all over the world for the treatment
and prevention of various ailments, particularly in developing countries where
infectious diseases are endemic and modern health care facilities are grossly
inadequate. In Africa, traditional medicine is of great value and more than
70% of the people refer to traditional healers concerning health issues (Kamanzy
et al., 2002). In Nigeria, as in other African countries, several
roots, leaves, fruits and barks of plants are used for different medicinal purposes;
some of which have been discovered in many researches to be rich in secondary
metabolites like tannins, alkaloids, flavonoids, phenols, steroids and volatile
oils which are responsible for their therapeutic activities (Cowan,
1999; Rabe and Vanstoden, 2000). The continued investigation
of the secondary plant metabolites has led to important breakthroughs in pharmacology
and has helped tremendously in the development of modern pharmacotherapeutics
in Africa and other parts of the world (Nwaogu et al.,
Cochlospermum tinctorium is a savannah plant found in fallow farms across
Northern Nigeria. It is a shrub that grows up to 10 m high. The leaves are alternate,
palmate lobed with stipules. The inflorescence consists of bright yellow flowers
that are regular or slightly irregular and borne in racemes or panicles. Fruits
are elongated 3-5 valve capsule containing seeds that are embedded in cotton
foam (Hutchinson and Dalziel, 1963). The plant is commonly
known as Rawaya (Hausa) and is a familiar herb in the traditional medicinal
preparations in Northern Nigeria, where decoctions of the whole roots are used
as remedy for gonorrhea, jaundice and gastrointestinal diseases (Mann
et al., 2003). Elsewhere, the roots are used as remedy for malaria
and schistosomiasis (Traore et al., 2006) and
are also found to have hepatoprotective effects (Diallo et
Earlier studies have indicated that triacylbenzenes and long chain volatile
ketones (arjunolic acid), apocarotenoids, tannins (ellagitannins) (Diallo
et al., 1991) and 3-O-E-p-coumaroylalphitolic acid (Ballin
et al., 2002) were all isolated from the roots of C. tinctorium.
Few pharmacological studies have been conducted on the plant. In this study,
we report the phytochemical, antibacterial and time kill studies of methanol
extract of C. tinctorium, with a view to gaining insight into its therapeutic
potentials in its local use for the treatment of gastrointestinal ailments in
MATERIALS AND METHODS
The roots of Cochlospermum tinctorium were collected from Dakace
village along Jos road, Zaria, Nigeria in July, 2006. The plant was authenticated
at the Herbarium of the Department of Biological Sciences, Ahmadu Bello
University, Zaria, Nigeria and voucher specimen (No. 2314) was deposited
Preparation of Plant Material and Extraction
The fresh roots of the plant were air dried, pulverized to fine powder
using a porcelain mortar and pestle. The pulverized material weighing
150 g was extracted exhaustively with methanol by simple percolation (cold
extraction) for one week. The extract was filtered and concentrated in
vacuo at 40°C using a rota vapor after which about 7.05 g of crude
methanol extract was obtained.
The extract was subjected to various phytochemical tests to identify the constituent
secondary metabolites using standard methods as described by Harborne
(1984), Sofowora (1993) and Trease
and Evans (1989).
Six Clinical Bacterial Strains
Escherichia coli, Klebsiella pneumoniae, Corynbacterium
ulcerans, Staphylococcus aureus, Proteus mirabilis and Shigella
dysentriae were obtained from the Department of Microbiology, Ahmadu Bello
University Teaching Hospital (ABUTH) Shika. The isolates were purified on nutrient
agar (OXOID) plates and characterized using standard microbiological and biochemical
procedures as described by Cowan and Steel (1974).
Determination of Antibacterial Activity
The antibacterial activity was carried out by utilizing the hole-in-plate
bioassay procedure as reported by Karou et al. (2006).
Pure culture of the organisms was inoculated on to Mueller-Hinton Agar (MERCK)
incubated for 24 h at 37°C. About 5 discrete colonies were aseptically transferred
with a sterile wire loop into a tube containing sterile normal saline (0.85%
NaCl) and were adjusted to a turbidity of 0.5 MacFarland standards. The suspension
was used to streak on the surface of Muller Hinton agar plates with sterile
swab. A sterile 6 mm diameter cork borer was used to make holes into the set
agar in petri dishes containing the bacterial culture. The wells were filled
with 500, 1000 and 2000 μg mL-1 concentrations of the extract.
Standard antibiotics ciprofloxacin (10 μg mL-1) and gentamicin
(10 μg mL-1) were used as reference or positive control. The plates
were incubated for 24 h at 37°C. All the tests were performed in triplicate
and the antibacterial activities were expressed as mean diameter of inhibition
zones (mm) produced by the plant extract.
Determination of Minimum Inhibitory Concentration (MIC)
Minimum inhibitory concentration was carried out using micro broth dilution
in accordance with National Committee for Clinical Laboratory
Standard (2006). Serial dilution of the least concentration of the extract
that showed activity was prepared using test tubes containing 9 mL of double
strength broth. The tests tubes were inoculated with the suspension of the standardized
inocula and incubated at 37°C for 24 h. MICs were recorded as the lowest
concentration of extract showing no visible growth of the broth.
Determination of Minimum Bactericidal Concentration (MBC)
Minimum bactericidal concentration was determined by aseptically inoculating
aliquots of culture from MIC tubes that showed no growth, on sterile nutrient
agar plates and incubating at 37°C for 48 h. MBC was recorded as the
lowest concentration of extract showing no bacterial growth.
Determination of Rate of Kill
The rate of kill was determined only for bacterial isolates most susceptible
to the extract. Inocula were grown over night at 37°C in Mueller Hinton
Agar (MHA). The over night broth was adjusted to a 0.5 McFarland standard
in nutrient broth and a further dilution was made by inoculating 200 μL
in glass flask containing 20 mL of sterile nutrient broth. The flask was
shaken and incubated for 90 min at 37°C to ensure that the organisms
were out of their lag phases and into their logarithmic phases.
After the 90 min initial incubation period, 20 μL of the culture
of each organism was inoculated into test tubes of nutrient broth containing
10 mL of MIC of the extract for each organism. Control was set up consisting
of two duplicate test tubes as negative control (without extract) and
positive control with 0.5 μg mL-1 of ciprofloxacin. All
test tubes were incubated at 37°C and samples sub cultured at 2, 4,
6, 8, 10, 12 and 24 h. Colony counts were performed by making serial dilutions
in physiological saline, plating 100 μL of each dilution on to MHA
and incubated for 48 h at 37°C. Viable counts were calculated to give
cfu mL-1 and kill curve were plotted with time against logarithm
of the viable count. Each experiment was performed twice on separate occasions.
RESULTS AND DISCUSSION
Results of the phytochemical screening revealed the presence of alkaloids,
flavonoids, tannins and cardiac glycosides, but saponin was found to be
absent (Table 1). Antibacterial activity of the extract
at 2000 μg mL-1 showed that S. aureus and S.
dysentriae exhibited the highest zone of inhibition (19.0 mm) whereas
K. pneumoniae and P. mirabilis had the lowest (11.0 mm)
(Table 2). The extract exhibited MIC at 100 μg
mL-1 against S. dysentriae, 500 μg mL-1
against S. aureus and C. ulcerans, 1000 μg mL-1
against E. coli and P. mirabilis and 1500 μg mL-1
against K. pneumoniae (Table 3). The MBC showed
that the extract was bactericidal against S. dysentriae at 500
μg mL-1, S. aureus and C. ulcerans at 1000
μg mL-1, E. coli and P. mirabilis at 1500
μg mL-1 and K. pneumoniae at 2000 μg mL-1
(Table 3). Time kill studies showed that there was no
viable S. dysentriae after 6 h exposure (Fig. 1).
However, S. aureus was completely killed after 10 h exposure to
the extract (Fig. 2). The killing of S. dysentriae
was more rapid than that of S. aureus (Fig. 3).
||Zone of inhibition diameter (mm) produced by extract
of C. tinctorium
-: No activity at that concentration
||MIC and MBC of C. tinctorium extract ( μg
*: MIC,ø : MBC, -: No growth,
+: Turbid, ++: Very turbid
||Killing of S. aureus by C. tinctorium
||Killing of S. dysentriae by C. tinctorium
||Comparison of killing of S. aureus and S.
The phytochemical screening of methanol extract of C. tinctorium showed
the presence of flavonoids, alkaloids, tannins and cardiac glycosides. These
metabolites have been shown to be responsible for various therapeutic activities
of medicinal plants (Trease and Evans, 1989). Flavonoids
especially are known to be effective antimicrobial agents against a wide array
of microorganisms; the activity is attributed to their ability to complex with
extra cellular and soluble proteins and with bacterial cell wall (Cowan,
The result of antibacterial activity showed that the extract was active on
all the tested microorganisms at different concentrations. The zones of inhibitions
were highest at 2000 μg mL-1 for all the organisms. These zones
were found similar to the zones produced by the standard antibiotics ciprofloxacin
and gentamicin at 10 μg mL-1 each (Table 2).
The MIC was lowest for S. dysentriae (100 μg mL-1) followed
by S. aureus and C. ulcerans (500 μg mL-1) and highest
for K. pneumoniae (1500 μg mL-1) (Table 3).
These findings are in agreement with the findings of Holetz
et al. (2002), however contradicts the findings of Suffredini
et al. (2006), which reported that their experience of screening
1200 plant extracts against four bacterial species showed that gram-negative
bacteria are hardly susceptible to plant extracts in doses as low as 200 mg
mL-1. The MBC result showed that the extract was bactericidal to
S. dysentriae at 500 μg mL-1, followed by S. aureus
and C. ulcerans (1000 μg mL-1) (Table 3).
Time kill studies on S. dysentriae (Fig. 1) and S.
aureus (Fig. 2) showed that the killings were time dependent.
It was also found that the killing of S. dysentriae was more rapid (after
6 h) than that of S. aureus (after 10 h) (Fig. 3).
Okemo et al. (2001) reported that MIC (4 mg mL-1)
of Azadirachta indica extract completely wiped out S. aureus in
6 h and noted that the killing was both dosage and time dependent. This suggests
that at higher concentration the organism would be killed at a faster rate.
The result obtained from the phytochemical screening has shown the presence
of some secondary metabolites. These compounds may be responsible for
the antibacterial activity of the plant extract. The activity was found
to be concentration and time dependent. These findings therefore support
the local use of C. tinctorium root extracts for treatment of
gastrointestinal, urinary tract and other infectious diseases in Northern
Nigeria. Further investigation using bioactivity guided fractionation
is currently going on to determine the active constituent(s) in our laboratory.
We are grateful to the authority of Ahmadu Bello University, Zaria for
providing the facilities for conducting this research and also to Mikhail
Sabo Abdullahi of the Department of Leather and Biotechnology, National
Research Institute for Chemical Technology (NARICT) Basawa, Zaria-Nigeria,
for technical assistance.
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