Isolation of Naphthoquinones from the roots of Euclea Natalensis and their in vitro antimycobacterial activity and toxicity

Abstract

Three naphthoquinones (shinanolone, 7-methyljuglone and diospyrin) were isolated from the dried roots ofEuclea natalensis, a tree belonging to the family Ebeneceae. The isolated compounds were purified by silica gel column chromatography and high performance liquid chromatography (HPLC). Nuclear magnetic resonance (1H NMR) was used to confirm their structure and purity. The percentage yield of shinanolone, 7-methyljuglone and diospyrin were 0.16, 0.12 and 0.32 respectively. Isolated compounds were tested for their antimycobacterial activity against drug-sensitive and multidrug-resistant clinical strains of M. tuberculosis by both the radiometric BACTEC 460 method and the colorimetric Alamar blue method. In the extracellular environment 7-methyljuglone was found to be the most active isolated compound which exhibited a minimum inhibitory concentration (MIC) in the range of „T0.5 ƒ®0.2 ƒÝg/ml. The extracellular MICs of the isolated compounds and the control (rifampicin) were in agreement in both the BACTEC 460 and the Alamar blue method. Furthermore, the cytotoxic effects of the isolated compounds were tested on human lymphocytes, fibroblasts and macrophages using the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay. 7-Methyljuglone was slightly toxic against resting and stimulated human lymphocytes (IC50 3.97 and 3.46 ƒÝg/ml, respectively), but not toxic against fibroblasts and macrophages. Rifampicin, crude extract, shinanolone and diospyrin were not toxic at tested concentrations. The compound that showed the highest activity in the extracellular environment was tested for intracellular activity on blood monocyte-derived macrophages and the THP-1 macrophage cell line using the radiometric BACTEC 460 method. 7-Methyljuglone exhibited antimycobacterial activity at „T5 ƒ®2.5 ƒÝg/ml in the intracellular environment. In clinical practice a combination of various drugs is necessary to prevent the emergence of resistance; therefore the in vitro activity of the most active naphthoquinone, 7-methyljuglone, was determined in two-drug combinations with standard antituberculous drugs (rifampicin, isoniazid, ethambutol and streptomycin) against M. tuberculosis using the radiometric BACTEC 460 method. In two-drug combinations, addition of sub-MICs of 7-methyljuglone, in both the extracellular and intracellular environment, resulted in a four-to six-fold reduction in MICs of rifampicin and isoniazid with fractional inhibitory concentrations (FIC) ranging between 0.25 and 0.5, suggesting a synergistic interaction against drug-sensitive strains of M. tuberculosis. The ability of 7-methyljuglone to inhibit the growth of drug-sensitive and multidrug-resistant strains of M. tuberculosis and the ability to enhance the activity of standard antituberculous drugs in vitro indicate that 7-methyljuglone may serve as a promising lead compound for future drug development for the treatment ofM. tuberculosis infections.