Plasmodium falciparum: Assessment of Selectivity of Action of Chloroquine, Alchornea cordifolia, Ficus polita, and Other Drugs by a Tetrazolium-Based Colorimetric Assay

A tetrazolium-based colorimetric selective assay (MTT-based CSA) was developed to assess the selectivity of antimalarial drugs. This in vitro assay, unlike all others, measures the ability of drugs to indirectly protect red blood cells (RBCs) from Plasmodium-falciparum-induced destruction. Optimum incubation time and number of cells needed were 5 days and RBCs per well, respectively. A parasitemia range of 0.375% to 3% was found to be suitable for this assay. The MTT-based CSA determined anti-P. falciparum strain DD2 activity of chloroquine at a higher 50% effective concentration (EC50) value (21.0？ g/mL) than the isotopic microtest (10.0？ g/mL). Artesunate and oxytetracycline achieved 90% effect against DD2 with minimal or no toxicity to RBCs. Against chloroquine sensitive strain 3D7, chloroquine and Alchornea cordifolia had EC50 values of 0.025？ g/mL and 4.9？ g/mL respectively, and selective index (SI) values of >2,000 and >69.4？ g/mL, respectively. 1. Introduction The threat of malaria has continued to plague millions of people living in most tropical countries despite numerous strategies to bring this disease under control. Chloroquine used to be the first drug of choice for the treatment of P. falciparum infections in Ghana and many African countries, but the emergence of drug-resistant strains has necessitated intensive search for new drugs [1–3]. Presently, Artesunate-Amodiaquine and Artesunate-Lumefantrine combinations are first-choice drugs in Ghana. The standard World Health Organization (WHO) method for testing new antimalarial drugs involves tedious microscopic parasite counting method (MPCM). Other in vitro methods are haem polymerization inhibition assay (HPIA) [4], lactate dehydrogenase assay (LDHA) [5, 6] and the isotopic microtest (IMT) [7]. All these in vitro tests measure some activity of the parasite and the endpoints are directly parasite related. Destruction of red blood cells (RBCs) is the end result of malarial infection. None of the available in vitro assays uses RBCs-related activity to determine endpoints. Furthermore, the available in vitro assays do not test for drug selectivity unless a separate assay on RBC toxicity is done. In this paper, we report on a tetrazolium-based colorimetric selective assay (MTT-based CSA) that uses indirect protection of RBCs from parasite destruction as an endpoint for measuring the effect of drugs on parasites and also determines the possible drug toxicity to the RBCs. 2. Materials and Methods 2.1. Drugs and Plant Extracts Chloroquine diphosphate (Sigma CQ) was obtained from SIGMA, St Louis,