Antibacterial Activity of the Volatile Oil of Phyllanthus muellerianus and Its Inhibition against the Extracellular Protease of Klebsiella granulomatis

Abstract

Aim: This study was carried out to evaluate the antimicrobial effect of the volatile oil of Phyllanthus muellerianus leaves against Klebsiella granulomatis and its inhibitory effect partially on the purified and characterized extracellular protease of this bacteria. Study Design: This involved the use of medicinal plants and pathogenic bacteria. Place and Duration of Study: Department of Biochemistry, Faculty of Science, Lagos State University, Ojo Lagos State Nigeria, between April, 2014 – September, 2014. Methodology: The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the hydrodistillated volatile oil against this bacterium were estimated by micro-dilution technique. The extracellular protease was partially purified with ammonium sulfate and Sephadex G-100 gel filtration and assayed using casein as substrate with the volatile oil as inhibitor. Results: The MIC and MBC obtained were 0.5% and ≥0.7% respectively. Gel filtration produced two peaks each for total protein and enzyme activity.  Highest fold of 4.67 was achieved with specific activity of 83.3 μmol/min/mg protein as compared to the crude enzyme. The oil competitively inhibited the partially purified extracellular protease of this bacterium with Vmax = 500 μmol/min and Km = 0.2 mg/ml (no inhibitor) and Km´ = 0.8 mg/ml (inhibitor). Optimal activities of this protease were obtained at pH 7.5 and 35°C.  None of the metallic chloride salts tested significantly increased the activity of the enzyme. However, Pb2+, Mn2+, Zn2+, K+, Hg2+, Co2+, Ba2+ and Mg2+ generally inhibited the enzyme activity. Conclusion: The volatile oil of Phyllanthus muellerianus possessed antimicrobial property and inhibited the extracellular protease of Klebsiella granulomatis. Further clarification and clinical verification of this oil will prove to be an effective match in combating burns and post-operative wound infections caused by Klebsiella granulomatis and similar species.