2.4. Antiplasmodial Activity of the Plant Extracts

The antiplasmodial activity of the extracts was assessed on cultured P. falciparum chloroquine-sensitive NF54 strain using the SYBR Green assay [15]. The extracts were weighed and dissolved in absolute methanol to obtain a stock solution of 100 mg/ml. The solution was then diluted with complete parasite media to a final working concentration of 10,000 μg/ml (10 mg/ml). The stock solutions were filter-sterilised through a 0.2 μm Millipore filters. Chloroquine was used as positive control. Two-fold serial dilutions of drugs (extracts) were performed to generate five concentrations for treatment of parasitized cells in vitro. One hundred microliters of P. falciparum malaria parasite culture suspension of NF54 (synchronized with 5% sorbitol to ring stage) was aliquoted into the wells of the pre-treated 96-well microtitre plate to a final haematocrit of 2% and parasitemia of 0.5%. Wells containing no drug but culture at the same parasitemia and haematocrit (with vehicle, i.e., distilled water) were included on each plate as negative control. Control wells also contained the vehicle and unparasitized red blood cells. Using the candle jar method, the plate was then placed in a humidified chamber in an incubator at 37°C for 72 h.

Evaluation of the outcome of the in vitro drug test was done by the SYBR Green method previously described [15]. Briefly, after 72 h of incubation of the parasite with the extract, 100 μl malaria SYBR Green 1 fluorescent (MSF) lysis buffer (containing 20 mM Tris-Cl (pH 7.5), 5 mM EDTA, 0.008% Saponin, 0.08% Triton-X 100), and SYBR Green were added to each well and mixed thoroughly. The plate was covered with aluminium foil and incubated at room temperature in the dark for at least 3 h. The SYBR Green fluorescence was read on a multiwell plate reader (Tecan Infinite M200, Austria) at excitation and emission wavelengths set at 497 nm and 520 nm, respectively. The experiments were performed in triplicate and each repeated at least once. The concentrations at which 50% inhibition of growth was obtained (IC₅₀ values) were determined by plotting the concentration of extract on x-axis against the percentage of inhibition on y-axis with dose response curves. Tables ​Tables11 and ​and22 contain two categories of antiplasmodial activities of plant extracts.

Categorisation of activities of plant extracts against Plasmodium falciparum.

Source [13].

Categorisation of biological substance of plant extracts based on antiplasmodial activity.

Source [11].