3.9. In Vivo Antimalarial Assay

The parasitemia of the donor mice was determined by preparing blood smears on microscope slides from blood film taken from the tails of infected mice [38]. The smear was fixed with methanol and stained with Giemsa to determine the parasitemia level of the donor under a microscope. When the parasitemia level was 30–40%, parasitized erythrocytes were collected from the donor mouse by cardiac puncture using a sterile syringe and placed in a Petri dish containing an anticoagulant (0.5% trisodium citrate) and then immediately diluted with uninfected mouse blood and normal saline (0.9%) in such way that the final volume contained 5 × 10⁷ infected erythrocytes/mL of blood [39]. The diluted blood (0.2 mL) was then injected into all the experimental mice intraperitoneally (IP) [40].

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].

Rane’s test, which evaluates the curative potential of RM-H1, was performed using the method described earlier [48]. Twenty-five mice were injected IP with an inoculum of 1 × 10⁷ P. berghei-infected erythrocytes on the first day (Day 0) [49]. At day 3, the animals were randomly divided into five groups with five mice in each group. Group 1 served as the negative control group and received the vehicle (2% Tween 80, 10 mL/kg/day), and Group 2 served as the positive control group and received chloroquine at 25 mg/kg/day. Groups 3–5 were treated with 8.75, 17.50, and 35.00 mg/kg/day RM-H1, respectively. Treatment continued for a further 3 days (i.e., 96, 120, and 144 h post-infection) [50]. Parasitemia levels were recorded daily throughout the experiment starting at day 3 [51]. PCVs, rectal temperatures, and body weights were measured just before the first dose (day 3) and at the end of the experiment (day 7). Thereafter, all groups were observed for 28 days, and their survival times were recorded [52].

Investigation of the prophylactic potential of RM-H1 was done following the method described previously [53]. Twenty-five mice were randomly assigned into five groups of five mice each. Group 1, which served as negative control received vehicle (2% TW80, 10 mL/kg/day) and Group 2 (the positive control group) received chloroquine 25 mg/kg/day. Groups 3–5 were treated with 8.75, 17.50 and 35.00 mg/kg/day of RM-H1, respectively_. Treatment was given orally for 3 days, 24 h after the last treatment (day 0), all mice were infected with an inoculum of 1 × 10⁷ P. berghei-infected blood [45,49]. Seventy-two h post-infection (day 3), blood smears were prepared from each mouse and the parasitemia level was determined [50]. PCV rectal temperature and body weight were measured just before parasite inoculation (day 0) and at the end of the experiment (day 3) [51]. Finally, the groups were followed for 28 days in order to record their survival time [52].