Comparative Antibacterial Studies On The Root, Stem Bark And Leaf Extracts Of Parkia Clappertoniana.
G Adeshina, O Onujagbe, J Onaolapo
Keywords
cold water extract, ethanolic extract., hot water extract, parkia clappertoniana
Citation
G Adeshina, O Onujagbe, J Onaolapo. Comparative Antibacterial Studies On The Root, Stem Bark And Leaf Extracts Of Parkia Clappertoniana.. The Internet Journal of Alternative Medicine. 2009 Volume 8 Number 2.
Abstract
The research on medicinal plants is gradually gaining popularity due to millions of people depending on the use of different parts of these materials for various ailments.The antibacterial activity of hot and cold water and ethanolic extracts of the root, stem and leaf of
Introduction
Many phytochemical constituents have isolated from Parkia species. Lemmich
Bacterial genera such as Staphylococci, Escherichia and Pseudomonads have been implicated in the above mentioned infectious diseases6.
Bacterial resistance to antibacterial drugs used in the treatment of some of the earlier mentioned infections has become a menace, therefore causing untold health challenges to patients. The aim of our study is to check the antibacterial effects of the root, stem and leaf of
Materials and Methods
Collection of plant materials: The plant
Extraction of plant materials: The root, stem bark and leaves were size reduced in order to facilitate drying. The plant parts were dried at room temperature in order to prevent loss of active constituents which may be thermo-labile and drying was continued until constant weight was obtained. Mortar and pestle were finally used to reduce the size of the dried plant parts to powder.
One hundred gram (100 gm) of powdered plant materials was weighed using an analytical balance and kept in the dessicator as to maintain a constant weight. This was transferred into a conical flask placed on a mechanical shaker and 250 ml of 99% ethanol was added and allowed to macerate for 6 hours after which it was decanted into a separating funnel and the filtrate collected. Another 250 ml of 99% ethanol was added to the residue, shaken and allowed to macerate for 6 hours. The total filtrate collected was evaporated to dryness by placing it on water bath at 50oC.
The hot and cold water extracts were obtained by the above procedure using 300 ml each of hot and cold distilled water. The dried extracts were kept inside dessicator before use.
Phytochemical analysis:
Phytochemical analysis was carried out on the plant extracts for the presence of chemical components such as, saponins, tannins, alkaloids, glycosides, flavonoids, triterpenoids, steroids and anthroquinones8.
Susceptibility testing:
Twenty milliliter (20 ml) of sterile nutrient agar was poured into the sterile petri-dish and allowed to set. The surface was flooded with 2 ml of 18 hour-broth culture of the test organism which has been standardized according to National Committee for Clinical Laboratory Standards9 by gradually adding normal saline to compare its turbidity to McFarland standard of 0.5 which is approximately 1.0 × 106 cfu/ml. The surface was allowed to dry and sterile No. 4 cork borer was used to bore six holes of about 2.5cm equal size on the surface. 0.1ml of the extract at different concentrations of 5%w/v, 10% w/v and 15% w/v was dropped into each hole and the plate was kept for about one hour at room temperature and incubated at 37oc for 18 hours. The diameter of zones of inhibition was measured after incubation to the nearest millimetre. The experiment was repeated three times and the mean diameter was taken.
Minimum Inhibitory Concentrations (M.I.C.) and Minimum Bactericidal Concentrations (M.B.C.) Determination.
The M.I.C. was determined by agar dilution method. Ten milliliter (10 ml) volume of double strength melted Mueller-Hinton agar at 45oC was diluted with equal volume of the test extract in graded concentrations. These were poured aseptically into sterile Petri-dishes and dried at 37oC for 1 hour with the lid slightly raised. Twenty microlitre (20 µl) of standardized test bacteria (106 cfu/ml) were aseptically inoculated on the sterilized paper discs placed on the agar surface at equidistance in triplicates for each concentration of the test plant extract10.
These were incubated at 37oC for 18 hours. The M.I.C. value was taken as the least concentration of the extract showing no detectable growth. Gentamicin was used as standard antibiotic.
The M.B.C was determined by transferring inoculated discs into a sterile 10ml recovery nutrient broth (Nutrient broth containing 3% v/v Tween 80) from the concentrations that showed no visible growth from the M.I.C. determination. These were incubated at 37oC for 72 hours. The least concentration of the extract that showed no bacterial growth in the recovery liquid medium was taken as the M.B.C.
Determination of rate of kill:
The rate at which hot water extract, which proved to be the most active of the tested extracts killed
A reaction mixture was made inside a conical flask containing 29 ml of sterile nutrient broth and 1.0 ml of the extract (15% w/v), with 0.1 ml of standardized overnight culture of the test organism. The mixture was shaken at 37oC and at various time intervals of 0, 30, 60 and 90 minutes, 0.1 ml of the mixture was taken using a micropipette, properly diluted and then plated on the surface of solidified sterile nutrient agar containing 3% Tween 80. It was then allowed to dry and plates were incubated at 37oC for 18 hours and the number of colonies were counted and recorded. This was repeated for 5% w/v and 10% w/v and gentamicin. A control was set containing nutrient broth and the test organism but without the extract. The test results were compared with that of the control.
Statistical analysis:
Results were expressed as mean ± standard deviation. The data was analyzed using Student’s t-test. P< 0.05 was considered significant and P>0.05 not significant.
Results
Phytochemical analysis:
The phytochemical analysis of the plant extracts revealed the presence of saponin, tannin, alkaloid, glycoside, flavonoids, triterpenoids, steroids and anthraquinones (Table 1).
Figure 1
Antibacterial susceptibility testing:
The result of the susceptibility test of the organisms to the extracts showed that the extracts had antibacterial activity against all the test bacterial isolates. The comparative study generally showed that the root, stem bark and leaf hot water extracts were more active than their ethanol and cold water extracts respectively. There was a statistical significant difference at P<0.05 between the antibacterial activities of the root hot water extract and the other root extracts, as the hot water extract showed a higher activity than them. The antibacterial activity of the stem bark hot water extract was higher against
The M.I.C. values are lower in the hot water extracts than ethanol and cold water extracts respectively (Table 5).
The rate by which the extracts kill the test organisms was progressive (Figures 1 - 3).
Figure 2
The results are expressed as mean ± standard deviation
Figure 3
The results are expressed as mean ± standard deviation
Figure 4
The results are expressed as mean ± standard deviation
Figure 5
Key : HWE – Hot Water Extract, CWE - Cold Water Extract, ETE - Ethanolic Extract, RT – Root, SB – Stem Bark, LF – Leaf
Figure 6
Figure 7
Figure 8
Discussion
The phytochemical screening of the plant parts extracts revealed the presence of saponin, tannin, flavonoid, anthraquinones, glycosides, triterpenoids, steroids and alkaloids. The presence of plant secondary metabolites in Parkia species, which are known to have broad spectrum of antibacterial activity have been reported11, 4, 2. El-Mahmood and Ameh2 reported the presence of phenolics in the root bark of
All the extracts showed antibacterial activity against all the tested bacteria species. This report disagrees with that of Ajaiyeoba11, who reported that ethanolic and water extracts of
The stem bark extracts showed more antibacterial activity than the other tested parts of the plant as revealed by this study. This result agrees with that of Millogo-Kone
The conventional antibiotic gentamicin, consistently showed superior antibacterial activity than the extracts similar to the results presented by other workers18, 2. This may be attributed to the fact that herbal medicinal products are prepared from plant and animal origins, most of the time subjected to contamination and deterioration while antibiotics are usually prepared from synthetic materials by means of reproducible manufacturing techniques and procedures19.
In conclusion,