Identification and pre-clinical characterization of medicinal plants used to prevent and cure malaria in pregnant women in Cameroon.

In the midst of transient victories by way of insecticides against mosquitoes or drugs against the parasitic disease, the most serious form of malaria, caused by Plasmodium falciparum, continues to be a major public health problem. Malaria in pregnancy compromises the health of both the mother and child and can be fatal for both. Particularly, malaria during pregnancy constitutes the major cause of low birth-weight and stillbirth in endemics countries. In Cameroon malaria is one of the major public health problems. The absence of an effective vaccine makes the parasitic disease very difficult to be eliminated and impossible to be eradicated. Treatment and prevention of malaria during pregnancy is currently a challenge for maternal health responsibles, due to the emergence of multi-drug resistant malaria parasites (e.g. to sulfadoxine-pyrimethamine used for IPT). However, there is, yet, very little and conflicting evidence on the safety of artemisinin-based combination treatments (ACTs) in particular during first term pregnancy. Traditional medicines have been employed to treat malaria for thousands of years. With the problems of increasing levels of drug resistance and difficulties in poor areas of effective antimalarial drugs being affordable and accessible, traditional medicines remains an important, sustainable source of treatment. Moreover, the development of evidence based safe and effective standardized herbal formulations for pregnant women with curative and transmission-blocking properties would be a major breakthrough to decrease malaria burden in women and their newborns. This Ph.D thesis aimed to a) identify medicinal plants used to prevent and cure malaria during pregnancy in Menoua West Cameroon; b) assess the activity of extracts from identified plants in-vitro against Plasmodium falciparum asexual blood-stages and gametocytes and against sporogonic stages of Plasmodium berghei in-vitro and in-vivo; c) estimate their cytotoxicity in vitro and d) design an evidenced based (safe and effective) plant combination (herbal remedy) with multistage effects on Plasmodium, suitable for women in pregnancy. Results show that 19 antimalarial plants are known to be employed in the study area to manage malaria during pregnancy and of these 11 were cited as commonly used. Of the latter 10 plants were possible to be collected (11 plant part materials) and 22 extracts were prepared (water and methanol). Seven, namely methanol and water extracts of D. edulis stem bark, methanol and water extracts of E. globulus leaves, P. americana stem bark and V. africana leaves methanol extracts, and methanol extract of C. citratus leaves were found to reduce by 50% in vitro asexual blood stage development at concentrations < 20μg/ml, on both P. falciparum parasite strains used. These results are in line with the one found in the literature. The stem bark methanol extract of P. americana was able to reduce the viability of mature gametocytes by 94% at the primary screening concentration of 100μg/ml. Its IC50 value was estimated to be 34.7 μg/ml. Comparing the anti-plasmodial activity of methanol and aqueous extracts of the tested plants, consistently higher activity was found in the former. Similarly, the methanol plant extracts were more active in vitro on the development of the sexual stages (early sporogonic development) that evolve in the mosquito vector. Two extracts, namely the stem bark methanol extract of P. americana and D. edulis, exhibited promising inhibitory effects against the insect transmissible stages of the parasite showing IC50 values in the range of 6 to 13 μg/mL in vitro and in vivo a reduction of oocyst numbers by more than 70%, after feeding on gametocytemic mice treated with 150 mg/kg. The results on transmission-blocking are promising since they are similar to the one obtain with the standardized herbal NeemAzal. Moreover, none of the 22 plant extract was found to have cytotoxic effects on normal human cell lines (NHF- A12-human dermal fibroblast and EA.hy926-endothelial). Interestingly, almost all the 22 extracts displayed some anti-tumor effects, reducing cell viability of melanoma and/or human breast cancer cells. Furthermore, the methanol stem bark extract of P. americana and D. edulis were found to possess some mosquitocidal potential when tested on the Sf9 insect cell line, but not as evident as NeemAzal® a azadirachtin A rich product used as reference. The findings of this doctoral research put in evidence antiplasmodial activity in about one-half of the tested medicinal plants against one or different life cycle stages of the parasite. Thus, based on this parasitological evidence, herbal remedies can be suggested to be used as a tool for the management of malaria in pregnant women. Further ethnobotanical studies including more villages and larger variety of respondents in the community will allow to identify additional medicinal plants uses, since our study was limited to a limited number of easy accessible villages. Moreover, our findings allow the design of rational combination formulations with antiplasmodial multi-stage activity. Preclinical studies will allow to assess efficacy and safety of such plant combinations, validating the concept of curative and preventive transmission-blocking herbal remedies.