3.9.2. 4-Day Suppressive Test (Peter’s Test)

A four-day suppressive test with mice infected with chloroquine-sensitive P. berghei was employed according to the method described previously [41]. Fifty-five mice were injected with an inoculum of 1 × 10⁷ P. berghei-infected erythrocytes IP on the first day (day 0) [42]. Two h post-infection, the mice were randomly distributed into eleven groups, each containing five mice. Group 1 served as a negative control group (received vehicle 2% Tween 80, 10 mL/kg/day) and Group 2 as positive control group (received chloroquine, 25 mg/kg/day). The remaining nine groups were treatment groups. Groups 3–5 received 100, 200, and 400 mg/kg/day of RM-M, respectively, while Groups 6–8 and Groups 9–11 received 8.75, 17.50, and 35.00 mg/kg/day RM-H and RMH-1, respectively. All test substances were administered orally using oral gavage, and the doses were determined based on the acute oral toxicity test results. The middle dose was one tenth of the safe dose (~2000 mg/kg for RM-M and ~175 mg/kg for RM-H and RM-H1). The higher dose was twice the middle dose, and the lower dose was half of the middle dose [43,44]. Treatment was started 3 h post-infection on day 0 and continued for an additional three consecutive days at 24, 48, and 72 h post-infection (until day 3). On day 4 of the experiment (at 96 h post-infection), blood was collected from the tail of each mouse, and a thin smear was prepared on a microscope slide to determine parasitemia [45]. In addition, body weight, rectal temperature, and PCV were measured just before infection and at the end of the experiment [46]. Afterwards, mice were observed for 28 days (day 0–27) to determine the MST for each group [47].