2.4.1. Four-Day Parasite Suppression Test

Peters' [28] protocol with slight amendments was exploited to complete the four-day parasite suppression test. P. berghei strain ANKA infected blood from the donor mouse was mixed with 1% (w/v) heparin. The infected blood was diluted in physiological saline to roughly 10⁸ parasitized erythrocytes per ml. The test animals were intraperitoneally injected with 0.2 ml and indiscriminately grouped into fives. The experimental groups were orally treated with 0.2 ml single dose of 125, 250, and 500 mg/kg of the test sample, 2 h after infection (D0) [29]. The negative and positive control groups were treated with 0.2 ml of the vehicle and 5 mg/kg of CQ, respectively. The mice were successively treated orally for 3 days (D1, D2, and D3) with corresponding doses. Thin blood smears were taken on the fifth day (D4) from tail snips, fixed in methanol, and stained with 10% Giemsa stain [30]. Parasitemia suppression was determined as per Tona et al.'s [27] formula:

where A is mean parasitemia in the negative control group on day 4 and B is parasitemia in the test group.

The mean survival time (days) for each group was determined over a period of 30 days after infection.