Phytochemical and Antibacterial Studies of Root Extract of Cochlospermum tinctorium A. Rich. (Cochlospermaceae)

INTRODUCTION

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., 2007).

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 al., 1992).

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 Northern Nigeria.

MATERIALS AND METHODS

Sample Collection
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 there.

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.

Phytochemical Screening

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).

Test Organisms
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).

Table 1:	Results of phytochemical screening

+: Present, -: Absent

Table 2:	Zone of inhibition diameter (mm) produced by extract of C. tinctorium

-: No activity at that concentration

Table 3:	MIC and MBC of C. tinctorium extract ( μg mL^-1)

*: MIC,ø : MBC, -: No growth, +: Turbid, ++: Very turbid


Fig. 1:	Killing of S. aureus by C. tinctorium extract


Fig. 2:	Killing of S. dysentriae by C. tinctorium extract


Fig. 3:	Comparison of killing of S. aureus and S. dysentriae

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, 1999).

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.

CONCLUSION

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.

ACKNOWLEDGMENTS

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.