Inhibition of biofilm formation in Mycobacterium smegmatis by Parinari curatellifolia leaf extracts.

BACKGROUND: Tuberculosis (TB) is a serious public health problem worldwide. Mycobacterium tuberculosis (M. tuberculosis) grows as drug tolerant pellicles. Agents that inhibit biofilm formation in M. tuberculosis have the potential to reduce the disease treatment period and improve the quality of tuberculosis chemotherapy. Parinari curatellifolia (P. curatellifolia) leaf extracts are claimed to treat symptoms similar to tuberculosis in ethnomedicinal practices. Mycobacterium smegmatis (M. smegmatis) is a surrogate organism used in antimycobacterial drug discovery assays. In this study, the effect of the leaf extracts of P. curatellifolia on M. smegmatis growth and biofilm formation was investigated in order to determine the basis of its use in traditional medicinal use. METHODS: Phytochemicals from P. curatellifolia leaves were prepared using a mixture of 50% dichloromethane (DCM): 50% methanol and by serial exhaustive extraction using different solvents of decreasing polarity. The solvents were used in the following order, hexane > dichloromethane > ethyl acetate > acetone >ethanol > methanol > water. The micro-broth dilution method was used as an antimycobacterial susceptibility test to screen for the extract that effectively inhibited M. smegmatis growth and biofilm formation. Biofilm quantification was performed by staining the biofilms with crystal violet and determining the amount of the stain using a spectrophotometer. In addition, the effects of combining the most active extract with kanamycin were also investigated. RESULTS: The minimum inhibitory concentrations (MIC) of the extracts were found to be 6.2 µg/ml for the acetone extract, 12.5 µg/ml for both the ethanol and the total extract and 50 µg/ml for both the methanol and ethyl acetate extracts. The ethanol extract, dichloromethane extract and water extract were the only extracts that effectively inhibited biofilm formation in M. smegmatis. Combining the ethanol extract with kanamycin enhanced the effect of the ethanol extract in terms of inhibition of biofilm formation. CONCLUSIONS: P. curatellifolia leaves contain phytochemicals that have the potential to be used both as antimycobacterial and anti-biofilm formation compounds.

Alkaloid extracts from Combretum zeyheri inhibit the growth of Mycobacterium smegmatis.

BACKGROUND: Current tuberculosis regimens have failed to combat the issue of drug resistance and ethno medicines may represent a possible source of antimycobacterial agents. Combretum species are well known in African traditional medicines and used for various ailments including pneumonia, venereal diseases like syphilis, mental problems, relief of sore throats and colds, fever, and chest coughs associated with tuberculosis. Alkaloids function as either hydrogen-acceptor or hydrogen-donor in hydrogen bonding critical for the interaction between targets thus, potentiating effects of curative agents on diseases. Alkaloid extracts from leaves of Combretum zeyheri, Combretum platypetalum, Combretum molle and Combretum apiculatum, were assessed for antimycobacterial activity to establish rationale for their use in traditional medicines for various ailments including pneumonia, relief of sore throats and colds, fever, and chest coughs associated with tuberculosis. METHODS: Alkaloids were extracted from the leaves of Combretum zeyheri, Combretum platypetalum, Combretum molle and Combretum apiculatum. The broth microdilution method was used for the screening of growth inhibitory activity. The standard drug rifampicin was used as the positive control. Alkaloid extracts from the most potent plant species, Combretum zeyheri were further investigated for time-kill dependency effects on drug transport in Mycobacterium smegmatis. RESULTS: Using the broth microdilution susceptibility method, C. zeyheri alkaloid extract, was found to have the most antimycobacterial effects with an MIC value of 125 µg/ml whilst MICs for C. molle and C. platypetalum were above 1000 µg/ml. An MBC value of 250 µg/ml was observed with alkaloid extracts from Combretum zeyheri whilst the remaining three Combretum species showed no bactericidal activity. It was also shown that C. zeyheri had potential efflux pump inhibitory activity. Determination of the time-kill kinetics of extracts from C. zeyheri showed not only a concentration-dependent activity but time-dependent bactericidal effect as well. CONCLUSIONS: Alkaloid extracts from the leaves of C. zeyheri have potential as a source of lead compounds that may be developed further into antimycobacterial compounds. The mechanism of action of may be due to inhibition of transport across the cell membrane. Further work needs to be done to isolate the active components in these extracts.