Isolation and characterization of the cytotoxicity, intracellular bioactivity and mechanism of antimycobacterial action of Euclea natalensis-derived naphthoquinones

The major cause of HIV-related mortality in Sub-Saharan countries is pulmonary tuberculosis (TB), which is an escalating threat due to the emergence of multidrug resistant (MDR) and extremely multidrug resistant (XDR) TB. There is clearly an urgent requirement for the identification of novel, affordable anti-TB (as well as anti-HIV) drugs. This study was undertaken with the objective of isolating and characterizing the antimycobacterial potential of 3 naphthoquinones, i.e. neodiospyrin, diospyrin and 7- methyljuglone present in the roots of Euclea natalensis. The laboratory research included: i) isolation of diospyrin and neodiospyrin, from the roots of E. natalensis; ii) assessment of the cytotoxicity of these agents and synthetic 7-methyljuglone for eukaryotic cells (Vero and THP-1 cells); iii) determination of the intracellular bioactivities of the naphthoquinones against the H37Rv strain of Mycobacterium tuberculosis (MTB); and iv) mechanistic studies designed to investigate the effects of the test agents on cation (K+/ Ca2+) fluxes and energy metabolism (ATP levels) in MTB and M. smegmatis. With respect to the first objective, the naphthoquinones (diospyrin and neodiospyrin) were isolated from crude methanol extracts of crushed roots using chromatography and spectroscopic analysis. The yields of the compounds were 0.16 %, 0.32 %, and 0.12 % for neodiospyrin, diospyrin (isolated from plant) and synthetic 7-methyljuglone (synthesised in laboratory), respectively. The effects of the compounds (0.3-50μg/ml) on the viability of Vero and THP-1 cells were measured using the XTT assay (sodium 3’-[1-(phenyl amino-carbonyl)-3, 4 tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic acid hydrate) based on cellular metabolic activity. All 3 test compounds were found to possess cytotoxic activity at 1.5- 12.5g/ml) for both cell lines. Intracellular bioactivity of the test agents was measured using MTB-infected THP-1 cells as a surrogate for infected human macrophages. Following exposure of the MTBinfected cells to the test naphthoquinones, at a concentration range of 6.25-25g/ml, for 5 days, the cells were lysed and the viability of MTB in the lysates was then measured using the BACTEC radiometric system. All 3 test agents were found to be bioactive intracellularly, with complete inhibition of growth detected at 12.5, 25, and 6.25g/ml in the case of neodiospyrin, diospyrin, and synthetic 7-methyljuglone respectively. The effects of the 3 naphthoquinones on mycobacterial cation fluxes were measured according to the magnitude of uptake of 86Rb+ (a surrogate for K+) and 45Ca2+, while ATP was measured using a chemiluminescence procedure. None of the test agents was found to affect Ca2+ uptake by the bacteria. However, all 3 test agents were found to be potent inhibitors of uptake of K+ by MTB and M. smegmatis, with inhibition detected at submicrogram concentrations of these agents. All 3 test agents, especially synthetic 7- methyljuglone, were found to interfere with energy metabolism in MTB, manifested as decreases in mycobacterial ATP levels. Synthetic 7-methyljuglone which has the lowest MIC value for MTB (0.5μg/ml), and which was the most potent inhibitor of energy metabolism in MTB, shows promise as a potential anti-TB agent.These agents also are of potential value in drug modelling, possibly in the design of novel anti-TB agents which selectively target mycobacterial K+ transporters.Thesis (PhD)--University of Pretoria, 2010.ImmunologyPhDUnrestricte