IN VITRO CHEMO-PREVENTATIVE ACTIVITY OF STRELITZIA NICOLAI ARIL EXTRACT CONTAINING BILIRUBIN

Background: The discovery of the only animal pigment, bilirubin, in the plant Strelitzia nicolai has triggered a vast number of questions regarding bilirubin’s formation and its role in the human body. Recent studies have confirmed that bilirubin at certain levels have many medical benefits. Various case studies have revealed that bilirubin is a potent antioxidant. Cervical cancer is one of South Africa’s largest womens’ health crises. It is estimated that it affects one out of 41 South African women and kills approximately 8 women in the country every day. Thus, the aim of this study was to investigate if the aril extract of Strelitzia nicolai (Regel and Körn.) containing bilirubin possesses anti-cancer activity and to determine its effect on the induction of apoptosis. Materials and methods: The DPPH activity was firstly used to determine the antioxidant effect of the extract. Thereafter, the cytotoxic effect was tested using the XTT assay. Apoptosis was confirmed and quantified using the Annexin V-PE kit and the morphology was studied using acridine orange and ethidium bromide. Results: The aril extract decreased cell viability by 52% and induced apoptosis in HeLa cells; as shown by the Annexin V-PE Apoptosis detection kit and morphological studies with acridine orange/ethidium bromide staining. Conclusion: The activity of the extract as a potent antioxidant was immensely enhanced as compared to the bilirubin standard. These results suggest that S. nicolai aril extract containing bilirubin works synergistically as opposed to bilirubin on its own. Furthermore, this extract might be a good candidate for the therapeutic intervention of cervical cancer

Investigation of the anti-mycobacterial mechanism of action of 7-methyljuglone

Although the naphthoquinone, 7-methyljuglone (7-MJ), is active against Mycobacterium tuberculosis (MTB) in vitro, neither the cellular site nor mechanism of anti-mycobacterial action of this agent has been identified. The primary objective of the current study was to investigate the mycobacterial outer membrane as a potential target of 7-MJ by measuring the effects of this agent (0.023 - 1.5 mg/L) on microbial ATP levels and uptake of K+. Methods: Bioluminescence and radiometric (uptake of 86Rb+) procedures were used to assay microbial ATP levels and K+ trans-port respectively. Results: Exposure of MTB (strain H37Rv) to 7-MJ for 60 min resulted in dose-related decreases in both microbial ATP levels and uptake of 86Rb+ which achieved statistical significance (P < 0.05) at concentrations of 0.4 and 0.1 mg/L respectively. Conclusions: These observations are compatible with the mycobacterial membrane as being the putative site of action of 7-MJ, targeting microbial energy metabolism and K+ transport.The National Research Foundation and the Medical Research Council, Pretoria, South Africa.http://www.SciRP.org/journal/ojrd)am2016ImmunologyPlant Scienc

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

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.ImmunologyUnrestricte

New insights into the presence of bilirubin in a plant species Strelitzia nicolai (Strelitziaceae)

Background: The fortuitous discovery of an animal pigment bilirubin found in the plant Strelitzia nicolai has opened an enormous number of questions regarding bilirubin’s formation and its ultimate function in the human body.Materials and Methods: A methodical review of bilirubin in humans and animals was carried out, information was gathered using published scientific journals, books and conference proceedings. Articles based on case studies of elevated levels of bilirubin were analysed thoroughly.Results: Even though for numerous years bilirubin was assumed to be merely a desecrate product of the heme catabolic pathway by greatest, and a likely lethal compound at worst; statistics from the last few decades clearly shows that placidly high serum bilirubin levels are robustly related to have abundant beneficial effects on the human body.Conclusion: This study reveals new insights into the presence of the only animal pigment found in Strelitzia nicolai arils, the potential advantages of bilirubin found in a plant and its therapeutic value indications. This review hopes to resuscitate researchers’ credence regarding bilirubin as a toxic compound.Keywords: bilirubin, heme, biliverdin, biliverdin reductase, Strelitzia nicola