Improvement of Secondary Metabolites from Phyllanthus odontadenius against Malaria by Mutagenesis

Aims: Majority of deaths in children aged under 5 years are due to Plasmodium falciparum malaria. Malaria deaths in children decreased but malaria remains a major killer of children, taking the life of a child every 2 minutes. This study aims to investigate the increasing of the in vitro antiplasmodial activities by mutagenesis techniques using gamma-rays (Cs-137) or sodium azide (NaN3) as mutagens. It will allow the importance of mutagenesis use as tools for improvement of secondary metabolites against malaria parasites using chemical or physical mutagens.

Study Design: Laboratory experiment tests : identification of plant material, immersion of seeds in SA (sodium azide) solutions or irradiation by Gamma-rays (Cs-137) of P. odontadenius seeds for improvement of secondary metabolites against malaria parasites, in vitro culture of seeds followed by the in situ culturing of plantlets for obtaining material of study, phytochemical screening of Phyllanthus odontadenius aerial parts to determine the change of compounds in comparison to controls, in vitro antiplasmodial tests for the determination of SA concentrations or those of gamma-rays doses which killing 50% of malaria parasite populations (IC50).

Place and Duration of Study: Department of Biotechnology and Molecular Biology, Department of Biochemistry: General Atomic Energy Commission, Regional Nuclear Studies Center of Kinshasa, P.O. Box. 868 Kinshasa XI, Democratic Republic of the Congo (DRC). National Institute of Biomedical Research (NIBR) at Kinshasa/Gombe (DRC). Laboratory of UMR-MD3, Aix-Marseille University, Faculty of Pharmacy/Marseille, France. The experiments were conducted from Junuary - December 2010; August and December 2011 and during May and September 2012 until July 2014.

Methodology: Aerial parts of plants M1 and M2 from Gamma-rays irradiation of P. odontadenius seeds or from immersion of P. odontadenius seeds in sodium azide solutions used as biological material for the in vitro antiplasmodial analysis. The in vitro antiplasmodial activities assays on clinical isolates of P. falciparum and on chloroquine-resistante P. falciparum strain K1 was determined using microscopic method, the isotopic micro-test method and using HRP2-based ELISA assay.

Results: Gamma-rays (Cs-137) increased (multiplied) the in vitro antiplasmodial activities from 2.48 up to 7.6 in comparison to control. Thus, the in vitro antiplasmodial activities were improved or exceeded from 147.57% up to 660% than those of control plant. SA had increased (multiplied) the in vitro antiplasmodial activities from 1.24 up to 10.15 comparing to the control plants. The antiplasmodial activities in vitro were exceeded compared to the control plants from 24.43% up to 915%.

Conclusion: Treatment of P. odontadenius seeds by Gamma-rays or by SA give plants high in vitro antiplasmodial activities. Values of in vitro antiplasmodial activities varried from 1.24 (147.57%) to 10.15 (915%). 125, 150 and 225 Gy of Gamma-rays (Cs-137) for physical mutagenesis and 10.15 and 17.5 mM of SA solutions for the chemical mutagenesis could be used for improving in vitro antiplasmodial activities against P. falciparum (clinical isolates of P. falciparum or chloroquine-resistant P. falciparum strain K1). Thus, plants extracts from treated seeds have justified the usefulness of mutagens in plant breeding particularly in the increasing production of secondary metabolite against malaria parasite.