In this study, the suppressive tests of extracts of the leaves of Acokanthera schimperi and Croton macrostachyus demonstrated a significant dose dependent chemosuppressive effect at various doses (200,400,600 mg/kg) for aqueous and methanol extracts. The highest percentage chemosuppression was exhibited by the aqueous extract of Croton macrostachyus, followed by the methanol extract of A. schimperi at 600 mg/kg.
Acute and sub-acute test results of the extracts of both plant species showed no sign of toxicity in all treated mice. The 4-day suppressive test is a standard test commonly used for antimalarial screening . Extracts of C. macrostachyus and A. schimperi produced significant chemosuppression in all treated groups.
Antiplasmodial activity has been related to a range of several classes of secondary plant metabolites including alkaloids, sesquiterpenes, triterpenes, flavanoids, limonoids, quassinoids, xanthones, quinines and phenolic compounds of which alkaloids have been the most important and have shown very interesting activities [8, 29]. Indeed, quinine is the first antimalarial drug that belongs to the class of alkaloids Croton spp. generally contain diterpenoids, triterpenoids, alkaloids, flavonoids, lignoids and proanthocyanidins , which have strong antiplasmodial activity. Therefore, the antiplasmodial activity observed in this study may be attributed to the presence of these bioactive compounds.
Both the methanol and aqueous extracts of C. macrostachyus exhibited comparable suppressive effect on P. berghei. However, aqueous extract of Acokanthera schimperi in all doses tested may be considered to have lower activity i.e. 22.34%, 22.72% and 24.60% parasitaemia reduction at 200, 400 and 600 mg/kg, respectively. On the other hand, methanol extract can be considered active with 34.8% parasitaemia reduction at 600 mg/kg and 31.8% at 400 mg/kg. Thus, the result of this study may justify the traditional use of the plant for antimalarial therapy in different parts of Ethiopia [13, 14].
Methanol and aqueous extract of C. macrostachyus and A. schimperi prevented body weight loss. Result of similar study on crude extracts and solvent fractions of Croton macrostachyus revealed the significant prevention of weight loss associated with increase in parasitemia level .
Hematological abnormalities are considered a hallmark of malaria . According to Iyawe and Onigbinde , Plasmodium berghei increases erythrocyte fragility and significantly reduces PCV in mice. Methanol and aqueous extracts of A. schimperi prevented PCV reduction in a dose dependent manner as compared to negative control group.
Another parameter used to evaluate the efficacy of antimalarial plant extracts in this study was mean survival time. A. schimperi significantly prolonged mice survival time compared to negative control group at 600 mg/kg and this could be attributed to parasitemia suppression effect of the extract of the plant. In contrast, all the doses of C. macrostachyus, which showed higher parasitemia suppression in this study, did not prolong mean survival time. This may show that half-life of the active compounds in plasma metabolism is shorter .
Comparatively, the methanol extract of the leaves of C. macrostachyus showed lower antimalarial activity than its water extract counterpart. On the other hand, water extract of the leaves of Acokanthera schimperi showed lower activity compared to that of methanol. During extraction, solvents diffuse into the solid plant material and solubilize compounds with similar polarity . This might indicate that the active compounds solubility which is responsible for the observed activity is different.
Chloroquine phosphate used in this study suppressed parasitaemia to non-detectable number, which is in agreement with  in which standard antimalarial drug cleared P. b erghei, to undetectable level. The results of the acute toxicity within 24 hours revealed that no toxic effect or mortality was observed in mice treated orally with methanol and aqueous extracts of Croton macrostachyus and Acokanthera schimperi as a single dose of 2000 mg/kg, which the single highest dose is recommended by OECD Guidelines 425 for testing acute toxicity. In addition, no changes in general appearance or behavioral pattern were noted until the end of 14 days. Therefore, absence of mortality up to an oral dose of 2000 mg/kg could indicate that the test extracts are safe and this could in turn explain the safe use of the plants by local people in Ethiopia who have been using them in traditional management of malaria [13, 14].
The median lethal oral dose (LD50) is greater than 2000 mg/kg if three or more animals survive . No death was observed in the animals receiving the extracts up to a dose of 2000 mg/kg body weight, which is about 10 times the minimum effective dose tested (200 mg/kg). If a test substance has LD50 higher than three times the minimum effective dose, it can be taken as a good candidate for further studies . The extracts from this study are, therefore, good candidates for further studies.
Repeated dose toxicity studies provide information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time  and repeated daily dosing is more clinically relevant than acute toxicity study. In this study, a 4-day administration of the extracts of 500, 750 and 1000 mg/kg daily revealed no sign of toxicity and all mice survived beyond the 30-days observation period.
According to Pillai et al. , the reduction in body weight is a simple and sensitive index of toxicity after exposure to toxic substances. In this study, there was no significant body weight change for aqueous and methanol extracts of A. schimperi and methanol extract of C. macrostachyus. Nevertheless, body weight loss in mice was observed at 1000 mg/kg in those treated with aqueous extract of C. macrostachyus. The decrease in body weight observed in the group treated with the highest dose could be attributed to the suppression of the animals’ appetite by the extract.
Investigation of the hematological parameters can also be used to determine the extent of deleterious effect of foreign compound including plant extracts on the blood constituent of an animal. The hematological parameters of treated mice (Hb and PCV of A. schimperi and PCV of C. macrostachyus), an index of anemia, did not show significant difference on day 4 compared to day 0. Similarly, WBC, in case of C. macrostachyus treated mice, which is an important index of pathological and physiological status , exhibited no significant difference. This effect of the extract on the hematological parameters of the animals might be an indication that it is unlikely to be toxic.