Phytochemicals and Antibacterial Evaluation of Root Bark Extract of Terminalia glaucescens

Article history: Received on: 13/05/2013 Revised on: 14/08/2013 Accepted on: 01/09/2014 Available online: 27/02/2014 The roots bark of Terminalia glaucescens was investigated for activity against four bacterial which are two grampositive and two gram-negative. Preliminary phytochemical screening of the root bark of Terminalia glaucescens revealed the presence of steroids, terpenoids, saponins, flavonoids, tannins and cardiac glycoside. The results of antibacterial test of the methanol crude extract and its fractions exhibited a wide range of activity against Staphylococcus aureus, Escherichia coli and Klepselle pneumonia at 200 mg/ml but there was no activity against Bacillus cereus. Fractions inhibited the growth of E. coli, S. aureus and K. pneumonia. The fractions had minimum inhibitory concentration (MIC) of 12.5mg/ml and minimum bactericidal concentration (MBC) of 25 mg/ml. The findings indicated that the extract and its fractions from root bark of T. glaucescens, contained bioactive components that have antibacterial properties.


INTRODUCTION
Medicinal plants have been in use for the eradication of human diseases since ancient times.In light of their established therapeutic efficiency, the pharmaceutical industries started to use crude extracts of medicinal plants for manufacturing drugs (Ali and Azhar, 2000).The acceptance of traditional medicine as an alternative form of health care and development of microbial resistance to the available antibiotics has led researchers to investigate the antimicrobial activity of medicinal plants (Lis-Balchin and Deans, 1996, Maoz and Neeman, 1998, Hammer et al., 1999).Presence of tannins, flavonoid, terpenoid, saponins , steroids, cardiac glycosides, volatile oils, alkaloids, anthraquinones and other phenolics have been reported to have antimicrobial activities (Hostettman and Nakanishi, 1979, Hostettman et al., 1995, Isaac and Chinwe, 2001).Terminalia glaucescens is belonging to family Combretaceae, is a tree up to 20 m high.It is commonly found in West Africa especially in Savannah regions.The plant is the most important medicinal species of the genus Terminalia (Ndukwe et. al., 2005), it is abundant in Nigeria.The plant is commonly called baushe (Hausa).
Six hundred gram (600g) of powdered root bark was extracted by soxhlet extractor used 1500ml of methanol as solvent at temperature of 85 °C, was concentrated using hot air sterilizing cabinet at 60 °C and yield 123.11gram of methanol crude extract.Split method of separation was adopted according to (Abubakar, 2009).The n-hexane was directly added to crude methanol extract and was vigorous stirring before filtration and the filtrate are all n-hexane soluble portion, which is the n-hexane fraction while the residue was allowed to dry and same method was repeated with ethyl acetate, n-butanol and finally the residue obtained is methanol fraction.N-hexane, ethyl acetate, nbutanol, and methanol fractions were obtained and were concentrated at 60 °C in hot air sterilizing cabinet.

Antibacterial assay
Bacterial cultures used in this research obtained from School of Medical Laboratory Science Department of microbiology, Usmanu Danfodiyo University, Sokoto.Bacterial isolates used in this study include; Escherichia coli, Staphylococcus aureus, Klebiella pneumonia and Bacillus cereus.All the cultures were grown on nutrient agar medium.
The plant extract & fractions (methanol extract, methanol fraction, ethyl acetate fraction, n-hexane fraction and n -butanol fraction) were tested for antibacterial activity by agar well diffusion assay.Concentration of 200mg/ml was prepared from each fractions and the extract.The concentration was used for antibacterial analysis using agar well incorporation method (Pelezer et al, 1993) plates of nutrients agar were prepared and allowed to solidify in petridishes, each plates were then seeded with test bacterium (standard size inoculums ,0.5 Mcfarland std).Five wells were made onto each of the agar plate with a sterile 6mm diameter cork borer.Each of the five wells were filled with a given concentration of methanol extract, methanol fraction, ethyl acetate fraction, n-butanol fraction and n-hexane fraction and gentamicin was used as control.The plates were then incubated at 37 °C for 24 hours.The diameters of zones of inhibition were measured using venier caliper and the value for each organism was recorded (WHO, 1997).

Minimum Inhibitory Concentration (MIC)
The method used in this was as described by (Irobi 1992), A 0.5 Mcfarland standard broth culture of the test organism, E. coli, S. aureus, and K. pneumoniae were prepared.Eight (8) test tubes were sterilized and set up on a test tube rack.Concentrations of 100mg/ml, 50mg/ml, 25mg/ml, 12.5mg/ml and 6.25mg/ml were prepared from the fractions.Test tube 6, 7 and 8 were set up as controls containing the un inoculated culture media, extract free growth medium and the test organisms without extract.Test tubes 1 -5 were inoculated with the test organism.
The mixtures were incubated at 37 °C for 24 hours.Turbidity was checked to determine MIC and recorded.The minimum inhibitory concentration of the fractions was regarded as the lowest concentration that shows no visible turbidity (growth of the test organism) (WHO, 1997).

Minimum Bactericidal Concentration (MBC)
To determine the MBC a wire loop full of the organisms were sub-cultured from the test tubes showing no turbidity onto sterile nutrient agar plate.The plates were incubated at 37 °C for 24 hours.The MBC is the plates showing 0 -5 colony forming unit (Richard, et al, 2004).

Statistical Analysis
Results are expressed as mean±standard error.The data obtained during zone of inhibition was subjected to one way analysis of variance (ANOVA), (triplet comparison) using least significant difference and t-test.The level of p<0.05 was considered significant.

RESULTS AND DISCUSSION Phytochemicals Screening.
The phytochemical screening of the root bark of T. glaucescens revealed the presence of tannins, saponins, flavonoids, terpenoids, cardiac glycosides and steroids, but alkaloids and anthraquinones are not detected as shown in Table 1.The presence of tannins, saponins, flavonoids, terpenoids, cardiac glycosides and steroids in plants part is an indication that the plant is of pharmacological importance (Hostettmann and Marston, 1995).

Anti-bacterial activity
The antibacterial activities of the methanol extract and its fractions of the root bark of T. glaucescens was tested against two gram positives and two gram negatives bacteria such as S. aureus, B. cereus and E. coli, K. pneumoniae respectively as shown in table 2. Methanol extract, n-hexane fraction, ethyl acetate fraction, n -butanol fraction and methanol fraction were active against or showed significant (p<0.05)inhibitions of S. aureus, E. coli, and K. pneumoniae but B. cereus was resistance to the extract, fractions and even the standard control (gentamicin) no doubt it has reported by (Richard et al 2004) that B. cereus is resistance to gentamicin, penicillin and other antibiotics.The plant extract and its fractions inhibited the growth of S. aureus, E. coli, and K. pneumoniae to a high degree and large zone of inhibition exhibited as shown in table 2. It means that the T. glaucescens can be use as broad spectrum anti-bacterial agent.Ndukwe et al, (2005) had reported that saponins, steroid and aglycones present in plant extract or fraction have varied uses as antiulcerogenic, anti-inflammatory, anti -edematous and analgesic.The activities of the extracts and the fractions from the root bark against S.aureus, which is the potential causative organism of the tooth decay and wound infections agreed with previous work (Rotimi and Bartlett, 1988) reported T. glaucescens as phytotherapeutic agent for dental hygiene and wound treatment.The observed antibacterial effects collaborate its traditional uses of the plant.The plant root bark is used traditionally in treatment of wound, fever, typhoid fever, dysentery, diarrhea, tooth decay, ulcers and various stomach related problems (oral communication with traditional healer).In this work the extract and fractions of the plant's root bark inhibited the growth of E. coli and K. pneumoniae to a high degree.These two bacteria are responsible for various stomach related illnesses, K. pneumonia is the causative organism of pneumonia and typhoid fever, a systemic infection associated with the consumption of contaminated food while, E. coli is responsible for a number of food related illnesses that manifest themselves in form of diarrhea, dysentery, fever etc.

Minimum Inhibitory Concentration (MIC)
The MIC results of the fractions were presented in table 3. The fractions inhibited and prevented their total growth of S. aureus, E. coli, and K. pneumoniae at MIC of 12.5mg/ml.The MBC results of the fractions shown in table 4, the fractions finally prevented the growth and killed the organisms completely at MBC of 25mg/ml.The findings in this work have justified the potent use of this plant in ethno medicinal treatment of oral infections of diarrhea, dysentery, fever, typhoid etc. which are caused by some of these organisms used in this study.However, this result strongly suggested that the antibacterial activity of the plant may due to the present of steroid, phenolics, saponins, tannins.

CONCLUSION
The findings in this work have justified the traditional use and the potent use of this plant in ethno medicinal treatment of oral infection of dysentery, fever, diarrhea, wound, tooth decay, malaria, ulcers, typhoid fever and various stomach related problems which are caused by some of these organisms used in this study.Work is still on in the isolate and characterization of the bioactive compounds in this plant.

Table 2 .
Zone of Inhibition at Concentration of 200mg/ml .