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Katsayal and Lamai Nig. Journ. Pharm. Sci., October, 2009, Vol. 8 No. 2, P. 121 – 125 simplex of which Phyllanthus niruri has been studied extensively and shown to possess antibacterial property (Taylor, 2003). The past few decades have experienced an overwhelming increase in global interest on the practice of traditional medicine and its use of medicinal plants to treat illness (Akarele, 1994). Plant-derived preparations and isolated phytochemicals or their model derivatives may be potentially useful to treat infectious diseases, especially in the light of the emergence of drug-resistant microorganisms and the need to produce more efficacious and cost-effective antimicrobial agents (Ncube et al., 2008). The use of antibiotics has revolutionized the treatment of various bacterial infections. However, their indiscriminate use has led to an alarming increase in antibiotic resistance among microorganisms (Hart and Karriuri, 1998). This necessitates the need for development of novel antimicrobials (Chopra et al., 1997). One way of preventing antibiotic resistance of pathogenic species is development of new compounds that are not based on existing synthetic antimicrobial agents (Rojas et al., 2006). The present study was therefore carried out to evaluate the preliminary phytochemical and antibacterial properties of the ethanolic extract of P. muellerianus stem bark, with the aimed of providing a lead compound(s) for the development of new, novel antibacterial agent(s). Plate 1: Phyllanthus muellerianus in its Natural Habitat MATERIALS AND METHODS Collection of Plant Material The plant, Phyllanthus muellerianus was collected from behind Ramat Hal, Ahmadu Bello University main campus in the month of February, 2009 and authenticated at the Herbarium Unit of the Department of Biological Sciences, Ahmadu Bello University, Zaria where it was assigned a voucher specimen number (925) by Mallam Umar Gallah. Solvent and Drugs and Test Organisms The solvent used for the extraction of the plant material is of Analar grade, and was purchased from Sigma-Aldrich representative in Nigeria. The standard drug used in this experiment, gentamicin, 122
Katsayal and Lamai Nig. Journ. Pharm. Sci., October, 2009, Vol. 8 No. 2, P. 121 – 125 was obtained from Lek Pharmaceutical Company Limited, Slovenia. The test organisms were obtained from the Department of Pharmaceutics and Pharmaceutical Microbiology, Ahmadu Bello University, Zaria. Processing of the Plant Material The stem bark was removed manually; air dried under shade and powdered using a wooden pestle and mortar. The preferable solvent used by the local people is mostly water and/or local gin; therefore, the extraction of the plant material was performed by weighing 100 g of the air dried, milled root-bark of Phyllanthus muellerianus and exhaustively extracted by repeated maceration with 70 % v/v aqueous-ethanol at room temperature. The alcoholic solutions were combined and evaporated to dryness at 45 °C under reduced pressure to obtain a dry extract which was weighed to yield 8.7 % and stored in a desiccator before used. Phytochemical Screening Phytochemical screening of the ethanolic extract was carried using standard methods (Evans, 2002; Sofowora, 2008) to screen for the presence of various chemical constituents. Test Organisms Standard type cultures of Bacillus subtilis (NCTC 10342), Escherichia coli (ATCC 11775), Pseudomonas aeruginosa (ATCC 10145) and Staphylococcus aureus (ATCC 021001) were kindly obtained from the Department of Pharmaceutics and Pharmaceutical Microbiology, Ahmadu Bello University, Zaria were used in this study. Standardization of Inoculum The bacterial cultures were prepared by transferring 2 to 3 colonies into bacteria growth medium (Luria broth agar) and incubated at 37° C for 14 hrs before use as described by Eloff (1998). The cells were maintained in Luria agar (10 g/l Bacto tryptone, 5 g/l Bacto yeast extract, 10 g/l NaCl at pH 7.0) in glass slant bottles. Antimicrobial Activity Determination Antibacterial screening was performed using an Agar diffusion method as described by Hugo and Russell, 1998. The plates were prepared in duplicate using nutrient agar. Three concentrations (10 mg/ml, 50 mg/ml and 100 mg/ml) of the extract in sterile water were prepared. Standard gentamicin, 2.5 mg/ml was employed as positive control. The plates were inoculated with standardized cultures of B. subtilis, S. aureus, E. coli and Ps. aeruginosa by flooding. Using the cup plate method described by Hugo and Russell (1998), a sterile cork borer was used to make 4 holes of about 8 mm in diameter into the solidified agar in each plate. 0.1ml of each concentration of the extract and the reference drug were applied in separate holes in each prepared plate. The plates were covered and kept for an hour to allow the extract and the standard drug to diffuse, before transferring to an incubator at 37° C for 18 hours after which zones of inhibition were measured to the nearest millimetre. RESULTS AND DISCUSSION The preliminary phytochemical screening revealed the presence of alkaloids, tannins, flavonoids, anthraquinones, saponins, steroids and reducing sugars (Table 1). Plants used in the treatment of disease are said to contain active principles which are phytochemicals with biological activity, some of which are responsible for the characteristic odours, pungencies and colours of plants while others give a particular plant its culinary, medicinal or poisonous virtues (Evans, 2002). From the result of the phytochemical screening, the extract was found to contain saponins, flavonoids and tannins which are well- 123
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