Evaluation of the antimicrobial activity of root and leaf extracts of Terminalia glaucescens.
Increases in the emergence of multiple resistant strains of clinically important pathogens have led to the search for more effective antimicrobial agents of plant origin with the aim of discovering potentially active ingredients to serve as a basis for the synthesis of new antimicrobials (Pretorius et al., 2003; Akinsulire et al., 2007). Terminalia glaucescens a plant indigenous to Africa belongs to the family Combretaceae. In Cameroon, it is traditionally used in the treatment of diabetes (Njomen et al., 2009). It is also one of the widely used plants employed as chewing stick in Nigeria thus various studies have been carried out on its antimicrobial activity against oral pathogens (Ogundiya et al., 2008). Terminalia species as reported by Rahman and Choudhary, (2005) are used in traditional medicines of Central African region. T. glaucescens is used in the treatment of dysentery, as an antimicrobial agent and has also found use in the last stages of AIDS (Koudou et al., 1995). Antiplasmodial activity ethanolic extract of the plant was described by Mustofa et al.,(2000).
The present study is designed to investigate the antimicrobial activity of the root and leaf extracts of T. glaucescens against selected bacteria and a fungal yeast with the aim of providing additional information on the antimicrobial activity of the plant.
Materials and methods
Collection and Extraction of Plant Materials:
The plant materials were collected from Ogbomoso, a town in Oyo State, South West Nigeria. The plant sample was identified by a botanist in the Department of Biological Sciences of Covenant University, where a voucher specimen was deposited.
The fresh leaves and roots of T. glaucescens were sun-dried and finely ground. A weighed quantity of the powdered leaves and roots was extracted with methanol by soxhlet extraction for 48hrs. The extracts were allowed to evaporate to dryness and stored in airtight bottles until ready for use.
Sources and Maintenance of Microorganisms:
Pure cultures of Gram positive organisms (Staphylococcus aureus, Bacillus anthracis), Gram negative organisms (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Proteus spp, Salmonella typhi) and a fungal yeast Candida albicans were obtained from the research laboratory of the Microbiology Department, Covenant University, Ota, Nigeria. The bacterial strains preserved on nutrient agar at 4[degrees]C were revived in Mueller Hinton broth and incubated overnight at 37[degrees]C. Bacterial suspensions were made to give approximately [10.sup.5] cfu/ml.
Antimicrobial Screening of extracts:
The antimicrobial activity of the extracts was determined using the agar well diffusion technique (Adeniyi and Ayepola, 2008) with modification. Mueller Hinton agar plates were seeded with 0.2mL of an overnight culture of each bacterial isolate. The seeded plates were allowed to set and a standard cork borer of 7mm diameter was used to cut uniform wells on the surface of the agar. The wells were then filled with 100[micro]L of each extract at a concentration of 50mg [ml.sup.-1] and 100mg [ml.sup.-1]. Ampicillin at 1mg [ml.sup.-1] was used as positive control. The plates were incubated at 37[degrees]C for 24h after which the diameter of zones of inhibition was measured in millimeters.
Determination of Minimum Inhibitory Concentration (MIC):
The determination of the minimum inhibitory concentration of the extract was carried out using the agar well dilution method as described by Adeniyi and Ayepola (2008).
Different concentrations of the extracts in the range of 50.0, 25.0, 12.5, 6.25, 3.125 and 1.56mg [mL.sup.-1] were prepared. 2mL of each dilution of the extract was mixed with 18mL of Mueller Hinton agar, poured into Petri dishes and allowed to set. The agar was streaked with an overnight broth culture of the bacterial isolates and incubated overnight. The plates were then examined for the presence or absence of growth. In all cases the lowest concentration at which there was no growth was recorded as the MIC.
Results and discussion
The results of the antibacterial activity of the root and leaf extracts of T. glaucescens are presented in Table 1. The antibacterial activity of the extracts as indicated by the zones of inhibition increased with increase in extract concentration. The diameter of the zones of inhibition ranged between 15mm and 33mm. The root extract at a concentration of 100mg [ml.sup.-1] was observed to have higher activity on all the test organisms than the leaf extract at the same concentration. The leaf extract was active on seven of the eight test bacteria. Bacillus anthracis showed the highest sensitivity to all the extracts with the highest zone of inhibition (33mm). Both the root and the leaf extracts had a high activity (30mm) on the fungal isolate Candida albicans at 100mg [mL.sup.-1]. The results of the minimum inhibitory concentrations (MICs) presented in Table 2 showed that the root and leaf extracts had the lowest MIC of 6.25mg [mL.sup.-1] on Pseudomonas aeruginosa.
The present study revealed that T. glaucescens extracts inhibited the growth of almost all test organisms but with varying effectiveness. The root extracts at 100mg [mL.sup.-1] inhibited the growth of all organisms while the leaf extracts at the same concentration inhibited the growth of seven out of the eight test organisms. Higher activity of the root extracts on the tested Gram positive organisms (Bacillus anthracis and Staphylococcus aureus) than the Gram negative organisms with the exception of Proteus spp was also observed. This is because Gram negative bacteria tend to have higher inherent resistance to antimicrobial agents as shown in similar reports by Ndukwe et al., (2005). Ogundiya et al., (2006) also reported a higher antibacterial activity of the root extracts of T. glaucescens against Staphylococcus aureus and Streptococcus pyogenes. Terminalia glaucescens is one of the plants used in the preparation of the "wonder cure" concoction used in the treatment of tuberculosis in Nigeria. The activity of the plant extract on Mycobacterium tuberculosis was confirmed by Adeleye et al., (2008). The antimicrobial activity of T. glaucescens as observed from this study may be attributed to the presence phytochemicals such as tannins, alkaloids, steroids, anthraquinones, flavonoids and saponins (Ogundiya et al., 2008; Adeleye et al., 2008). The results of the determination of Minimum Inhibitory Concentrations (MICs) showed the root and leaf extracts to have the lowest MIC of 6.25 on Pseudomonas aeruginosa an organism known for its intrinsic resistance to various antibiotics and was also found to be resistant to ampicillin used as a positive control in this study.
The results of this study confirms the potential uses of T. glaueeseens as a source of antibacterial agents against infections caused by Pseudomonas aeruginosa, Bacillus anthracis, Klebsiella pneumoniae, Escherihia coli, Salmonella typhi, Staphylococcus aureus, Candida albicans and Proteus spp.
Results from the present study have shown T. glaucescens as a potential source of antimicrobial drug against various pathogenic organisms. This is particularly important in combating the recent trend in the emergence of multiple drug resistant organisms. Further studies are however necessary for the development of new antimicrobials from this plant.
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Department of Biological Sciences, College of Science and Technology, Covenant University, PMB 1023, Ota, Ogun State, Nigeria.
Corresponding Author: Olayemi O. AYEPOLA, Department of Biological Sciences College of Science and Technology, Covenant University, PMB 1023, Ota, Ogun State, Nigeria. Tel. No.: +234 803 478 5269
Table 1: Antimicrobial activities of the root and leaf extracts of T. glaucescens at 50mg [mL.sup.-1] and 100mg [mL.sup.-1] Organisms TGR 50mg [mL.sup.-1] TGR 100mg [mL.sup.-1] Pseudomonas aeruginosa 28 30 Bacillus anthracis 30 33 Klebsiella pneumonaie 22 25 E. coli -- 20 Salmonella typhi 20 25 Staphylococcus aureus 30 32 Candida albicans -- 30 Proteus spp 30 32 Organisms TGL 50mg [mL.sup.-1] TGL 100mg [mL.sup.1] Pseudomonas aeruginosa 15 30 Bacillus anthracis 15 25 Klebsiella pneumonaie -- 16 E. coli -- -- Salmonella typhi 20 20 Staphylococcus aureus 15 24 Candida albicans 22 30 Proteus spp 15 22 Organisms Positive control Pseudomonas aeruginosa -- Bacillus anthracis -- Klebsiella pneumonaie 20 E. coli Salmonella typhi 35 Staphylococcus aureus 30 Candida albicans -- Proteus spp -- Resistance = no zone of inhibition, Positive control = Ampicillin (1mg [mL.sup.-1]) TGR = T. glaucescens root extract, TGL = T. glaucescens leaf extract Table 2: Minimum Inhibitory Concentration (MIC) of the root and leaf extracts of T. glaucescens Organisms TGR (mg [mL.sup.-1]) TGL (mg [mL.sup.-1]) Pseudomonas aeruginosa 6.25 6.25 Bacillus anthracis 25 50 Klebsiella pneumoniae 50 100 Escherichia coli 25 6.25 Salmonella typhi 25 50 Staphylococcus aureus 25 50 Candida albicans 50 50 Proteus spp 50 50 TGR = T. glaucescens root extract, TGL = T. glaucescens leaf extract