|Year : 2012 | Volume
| Issue : 1 | Page : 58-62
Antimicrobial potential of Dialium guineense (Wild.) stem bark on some clinical isolates in Nigeria
FA Olajubu1, I Akpan2, DA Ojo1, SA Oluwalana3
1 Department of Medical Microbiology and Parasitology, Olabisi Onabanjo University Teaching Hospital, Sagamu, Nigeria
2 Department of Microbiology, University of Agriculture, Abeokuta, Nigeria
3 Department of Forestry and Wildlife, University of Agriculture, Abeokuta, Nigeria
|Date of Web Publication||1-Jun-2012|
F A Olajubu
Department of Medical Microbiology and Parasitology, Olabisi Onabanjo University Teaching Hospital, Sagamu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context : The persistent increase in the number of antibiotic-resistant strains of microorganisms has led to the development of more potent but also more expensive antibiotics. In most developing countries of the world these antibiotics are not readily affordable, thus making compliance difficult. This calls for research into alternative sources of antimicrobials. Dialium guineense is a shrub of the family Leguminosae. Its stem bark is used for the treatment of cough, toothache, and bronchitis. Aims : Despite the acclaimed efficacy of D guineense, there is no scientific evidence in its support. This work was carried out to assess the antimicrobial activity of D guineense in vitro against some clinical isolates. Materials and Methods : D guineense stem bark was collected and 50 gm of air-dried and powdered stem bark of the plant was soaked for 72 hours in 1 l of each of the six solvents used in this study. Each mixture was refluxed, agitated at 200 rpm for 1 hour, filtered using Whatman No. 1 filter paper and, finally, freeze dried. The extracts were then tested for antimicrobial activity using the agar diffusion method. Results : The highest percentage yield of 23.2% was obtained with ethanol. Phytochemical screening showed that D guineense contains anthraquinone, alkaloids, flavonoids, tannins, and saponins. The antimicrobial activity of the extracts revealed a broad spectrum of activity, with Salmonella typhi and Staphylococcus aureusa showing the greatest zones of inhibition (18.0 mm). Only Candida albicans among the fungi tested was inhibited by the extract. The greatest zone of inhibition among the fractions was 16.0 mm. D guineense exhibited bactericidal activity at the 7th and 9th hours against Streptococcus pneumoniae and S. aureus 25923 while the 10th hour against S. typhi and C. albicans. The greatest activity was noted against S pneumoniae, where there was reduced viable cell count after 6 hours of exposure. Conclusion : Stem bark extract of D guineense (Wild.) has the potential to be developed into an antimicrobial agent
Keywords: Dialium guineense , antimicrobial activity, broad spectrum, clinical isolates
|How to cite this article:|
Olajubu F A, Akpan I, Ojo D A, Oluwalana S A. Antimicrobial potential of Dialium guineense (Wild.) stem bark on some clinical isolates in Nigeria. Int J App Basic Med Res 2012;2:58-62
|How to cite this URL:|
Olajubu F A, Akpan I, Ojo D A, Oluwalana S A. Antimicrobial potential of Dialium guineense (Wild.) stem bark on some clinical isolates in Nigeria. Int J App Basic Med Res [serial online] 2012 [cited 2020 Jan 22];2:58-62. Available from: http://www.ijabmr.org/text.asp?2012/2/1/58/96811
| Introduction|| |
The oldest form of health care known to mankind involves the use of plants and their parts for therapeutic purposes, a practice that has been embraced by all cultures throughout the world.  The World Health Organization estimates that about 80% of the world's population uses herbal medicine for some aspect of their health care.  Despite the popularity of modern medicine and the variety of drugs available for various ailments, it has been observed that 85% of patients combine herbal therapy with the medicines prescribed at hospitals or clinics.  This shows the level of confidence patients have in herbal recipes. The situation in the United States is not any different, and it has been noticed that 25% of prescription drugs dispensed in the US contain at least one active ingredient derived from plant material. 
The persistent increase in antibiotic-resistant strain of microorganisms has led to the development of more potent but also more expensive antibiotics, such as the third-generation fluoroquinolones and the cephalosporins. ,, In most developing countries of the world, these antibiotics are not readily affordable, which makes compliance difficult. This calls for research into alternative sources of antimicrobials. Dialium guineense is a shrub of the family Leguminosae. It has a straight, grayish and smooth stem. The stem bark is used for the treatment of cough, toothache, and bronchitis.  Despite its acclaimed efficacy, there is as yet no scientific in its support. This work was carried out to assess its in vitro antimicrobial activity against some clinical isolates.
| Materials and Methods|| |
Plant collection and authentication
D guineense stem bark was collected with the assistance of a herbal practitioner at Ode-Lemo, Ogun State of Nigeria. A sample of the plant was taken to the Forest Research Institute of Nigeria (FRIN) Ibadan, Nigeria, for further confirmation and a voucher specimen was deposited there [Forest Herbarium, Ibadan (FHI) No. 108009].
A 50-g portion of air-dried, powdered, stem bark of D guineense was soaked in 1 l of each of the six solvents used [i.e., sterile distilled water, absolute ethanol, ethanol 50% (v/v), chloroform, petroleum ether, and acetone] for 72 hours according to the method of Rojas et al.  Each mixture was refluxed, agitated at 200 rpm for 1 hour, and filtered using Whatman No. 1 filter paper. Following this, the aqueous filtrates were freeze dried, while the alcohol filtrates were placed in a vacuum oven at 40°C and dried for 3 days to obtain the dry extracts. The percentage yield and the pH of the extracts in the different solvents were determined. All crude extracts were stored at 4°C until they were needed for use.
Qualitative chemical analysis of the plant was carried out using the standard methods described by Treas and Evans. 
Fractionation of the crude extract
Forty grams (40 g) of the crude extract (coded CEDG), was adsorbed onto silica and allowed to dry before it was packed on the Buchner funnel column of Kieselgel 60 (ASTM 70-230 mesh). The elution was effected with n-hexane, dichloromethane, ethyl acetate, acetone, and ethanol successively. The extracts were in turn concentrated to dryness in a vacuum oven. The resultant fractions were then tested for antimicrobial activity using the agar diffusion method.
TLC was done on bioactive fractions using analytical silica gel 60 GF 254+366 precoated aluminum-backed plates (Merck; 0.25 mm thick). One milligram of the sample was dissolved in 2 ml of methanol and spotted on a silica gel sheet and developed using an ethyl acetate: acetone (9:1) solvent system. The resulting spots on the chromatography paper were visualized under ultraviolet (UV) light (254 nm) and detected using 10% H 2 SO 4 in methanol spray reagent. Fractions having the same TLC patterns were bulked together and concentrated in vacuo, resulting in four pure fractions. These were further subjected to antimicrobial activity testing.
Assay of antimicrobial activity
The microorganisms used for the assessment of the antimicrobial activity of the plant extracts were procured from the Nigerian Institute of Medical Research and Olabisi Onabanjo University Teaching Hospital. Different strains were also used [Table 1].
|Table 1: Characteristics of microorganisms used to assess the antimicrobial activity of the plant extracts|
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The method of McFarland was modified for the preparation of the inoculum. The optical densities of the 0.5 McFarland turbidity standard and the organism suspensions were compared and further adjusted on a UNICO® 2100 spectrophotometer at 520 nm wavelength.
The agar well diffusion method described by Shahidi and Rashidi  was adopted for antibacterial activity assessment.
Müller-Hinton (MH) agar (Fluka Bichemika, Lot 1198898, Spain) and heated blood agar (HBA) were used for the bacterial isolates, while Sabouraud dextrose agar (Lab M Batch No. 053741, UK) was used for the fungal isolates. Agar plates were seeded with 2.0 ml of the bacteria preparation. Any excess suspension was drained off using a pad of filter paper. Wells of 6-mm diameter were bored in the MH and HBA culture media with a sterile cork borer. A concentration of 100 mg/ml of the extract was prepared in the appropriate solvent and 0.2 ml of the extract was used to fill each well. The plates were incubated at 37°C for 24 hours. The diameters of zones of inhibition were measured in millimeters. All samples were tested in duplicate and the average was recorded as the mean inhibition zone. Gentamycin at 1 μg/ml and the various extractants served as positive and negative controls, respectively.
For screening the antifungal activity of the extract, the method described by Awoderu et al. was adopted. Incubation was done at 30°C for 48 hours, and griseofulvin and the extractants served as positive and negative controls, respectively.
Determination of minimum inhibitory concentration and minimum bactericidal concentration of the extract
Starting from the initial concentration of 100 mg/ml, the plant extract was serially diluted with molten Müller-Hinton agar to achieve final concentrations of between 10 mg/ml and 0.63 mg/ml and poured aseptically into sterile Petri dish More Detailses as described by Musa et al. These agar-plant extract mixtures were allowed to set. Sterile paper discs (Whatman No. 1 filter paper, 5 mm in diameter) were firmly placed on the agar surface. Twenty microliters (20 μl) of freshly standardized bacteria cultures were then dispensed on the paper disc. The plates were incubated at 37°C for 24 hours. The lowest extract concentration that inhibited the growth of the test organisms was taken as the MIC. The paper discs in the MIC determination that showed no visible growth around their edges were placed in 5 ml glucose broth and incubated for 24 hours and then subcultured on freshly prepared 10% blood agar (BA). The BA plates were incubated at 37°C for 24 hours. The lowest concentration of the plant extract at which no growth was observed on the cultured plates was taken as the MBC of the extract.
Kinetics of bactericidal activity of the extract
Twenty milliliter (20 ml) of the MBC of plant extract was prepared with sterile normal saline using a modification of the method described by Olonitola et al. The extract was then inoculated with 20 μl of 1.5 × 10 8 cfu/ml of some selected isolates that had exhibited sensitivity to the extract, namely, Staphylococcus aureus 25923, Streptococcus pneumoniae, Salmonella More Details typhi, and Candida albicans. At different time intervals (0, 1, 2, 3, …12 h), 20 μl placed on five different spots of a freshly prepared plate count agar and Sabouraud dextrose agar (SDA), which were then incubated at 37°C and 28°C, respectively, for 24 hours. The average of the five plate counts was recorded to the nearest whole number.
| Results|| |
The colors of the crude extracts were between light brown in isopropanol to brownish-black in chloroform. The highest percentage yield of 23.2% was obtained with ethanol, while the lowest yield of 0.2% was obtained with petroleum ether [Table 2].
The phytochemical screening showed that D guineense contains anthraquinone, alkaloids, flavonoids, tannins, and saponins. The antimicrobial activity of the extracts was revealed by a broad spectrum of action, with S typhi and S aureus a showing the highest zone of inhibition (18.0 mm). Only C albicans, among the fungi tested, was inhibited by the extract. The highest zone of inhibition with the fractions was 16.0 mm [Table 3].
|Table 3: Antimicrobial activities of D guineense crude extracts expressed as inhibition zones (in millimeters)|
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The MIC of D guineense crude extract against Escherichia More Details coli 25922, Bacillus subtilis, and S typhi was 0.63 mg/ml, while the MBC was 5.0 mg/ml against S typhi and C albicans [Table 4].
|Table 4: Minimum inhibitory concentrations and minimum bactericidal concentrations of the ethanolic extract of D guineense (in mg/ml) and standard drugs (in μg/ml) on sensitive bacteria isolates|
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D guineense exhibited bactericidal activity at the 7 th and 9 th hours against Streptococcus pneumoniae and S. aureus 25923 while the 10 th hour against S. typhi and C. albicans. The greatest activity was noted against S pneumonia, with drastically reduced viable cell count after 6 hours of exposure.
Fractionation by TLC produced four fractions, which were further tested for antimicrobial activity. The DGEF1 fraction of the ethanolic extract showed the maximum activity [Table 5].
|Table 5: Activity of pure fractions from D guineense extracts on some organisms, expressed as zone of inhibition (in millimeters)|
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| Discussion|| |
The ethanolic extract of D guineense was the most active against the microorganisms studied. This finding agrees with that of some other studies , and shows that preparation of this herb in tinctures is the best method for extracting and concentrating the active components of the plant. The use of alcohol as an extractant will also enhance the preservation of the extracts and its assimilation by the body.
Anthraquinone which has been known to form an irreversible complex with nucleophilic amino acids, leading to their inactivation and loss of function, was found in D guineense extract and might be responsible for its broad-spectrum antimicrobial activity.  The crude extract of D guineense was able to inhibit multiple drug-resistant strains of Pseudomonas aeruginosa up>a. This agrees with the work of Nascimento et al., who demonstrated that P aeruginosa that was resistant to many antibiotics was inhibited by extracts from clove, jambolan, and thyme.  This puts D guineense in the ranks of the potential novel antimicrobials that may help solve the current problem of bacterial drug resistance.
The effectiveness of an antibacterial agent is measured by its ability to inhibit or kill bacteria.  The MIC and the MBC of D guineense were 0.63 mg/ml and 5 mg/ml, respectively; though these values are higher than that of the control antibiotic gentamycin, they can still be considered reasonably good. Our findings are in agreement with the work of Akinpelu and Onakoya.  D guineense demonstrated bactericidal activity against S aureus a, S pneumoniae, S typhi, and C albicans, which justifies its use in the treatment of enteric and respiratory infections. , The reduction in antimicrobial activity of the fractions versus the crude plant extract (as revealed by the smaller inhibition zones) might be an indication that other components in the plant, though not antimicrobial in nature, might contribute to the overall performance of the whole plant.
Studies are ongoing to ascertain the mode of antimicrobial activity of this plant, which has the potential for being developed into a novel antimicrobial drug.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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