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Box , Eldoret, Kenya. This study was carried out by Jackie K. Obey and is part of her doctoral thesis; all the data are available from her upon request. There is an increasing need for innovative drug and prophylaxis discovery against malaria.
The aim of the present study was to test in vivo antiplasmodial activity of Croton macrostachyus H. Euphorbiaceae stem bark extracts from Kenyan folkloric medicine. The study showed that it is possible to inhibit the growth of the parasites by various stem bark extracts of C. Medicinal plants have been used to cure parasitic infections from time immemorial. The number of drug-like molecules possibly present in the vast amount of species fungi, bacteria, marine invertebrates, and insects has been estimated to exceed 10 60 [ 1 ].
As a source of novel drugs, plants remain grossly understudied and underused, especially in the developed world [ 2 , 3 ]. In China, India, Africa, and Latin America, modern drugs are not available, or, if they are, they often prove to be too expensive, unavailable, or inaccessible. Malaria has historically been among the most deadly parasitic infections in many tropical and subtropical regions [ 4 , 5 ].
Plasmodium falciparum is the most important agent of human malaria, transmitted by the Anopheles mosquito into the human blood. Worldwide million malaria cases were reported in Until recently, malaria used to be the leading cause of death among children in sub-Saharan Africa [ 5 ]. The improvements can be attributed to WHO-recommended core interventions, vector control, chemoprevention, diagnostic testing, and treatment, which all have proven to be cost-effective.
Emerging parasite resistance to antimalarial medicines as well as mosquito resistance to insecticides could render some of the current tools ineffective and trigger a new rise in global malaria mortality. The resistance of P. Except antifolate antimalarial drugs other commonly used antimalarial agents are based on plant-derived compounds, quinine, and artemisinin derivatives, which remain vital drugs in the treatment of malaria [ 10 , 11 ].
Croton macrostachyus Hochst. The bark, fruits, leaves, roots, and seeds of C. In the distribution area there is a high degree of medicinal use consensus for bleeding, blood clotting, cancer, constipation, diarrhea, epilepsy, malaria, pneumonia, purgative, ringworm, skin diseases or infections, stomach ache, typhoid, worm expulsion, and wounds [ 13 ].
Leaf decoction, infusion, or maceration, stem or root bark, and leaf sap of C. Members of the genus Croton and different parts of plant have been used traditionally to treat infectious diseases such as measles and typhoid fever in Kenya [ 14 ] and against malaria in Ethiopia [ 15 ]. Antimalarial activity against Plasmodium berghei in mice has been found from C. In the present study antiplasmodial activity of C. The stem bark extracts were prepared as described before [ 19 ]. Shortly, the fresh stem bark was cut into small pieces using a pen knife.
The cut bark was air-dried in a shaded area for three weeks. The air-dried bark was powdered using a mechanized hand grinder. The soaked extract was separated from the plant residue by using a Buchner funnel. Plasmodium berghei strain ANKA, a chloroquine-sensitive strain of malaria parasite, was used in the in vivo study. The percent parasitemia and the erythrocytes were counted using the white blood cell count method. The test animals were infected intraperitoneally with 0.
An acute toxicity test was carried out to ascertain the safety of the extract. For the test groups an oral dose of 0. The negative control group received 0. The weights of all animals were taken before and after the experiment. The animals in each group were observed for any change in physical activity and signs of abnormal growth or disease condition.
This included observations of mortality, hair erection, tremors, lacrimation, convulsions, salivation, diarrhea, and abnormal features in organs and blood. The study design was a quantitative case control study described by Peters in [ 20 ]. Consent to use the experimental animals in the study was obtained from the ethical committee of the Kenya Medical Research Institute, Center of Biotechnology and Research Development, Department of Malaria.
Each mouse was infected intraperitoneally by injecting 0. Each study group included PC and NC groups. On days 0, 1, 2, and 3 the animals were treated once orally with the different doses of the extracts in a volume of 0. The survival of the mice in all the groups was checked twice a day. Parasitized erythrocytes RBC were counted in Giemsa stained thin films from tail blood on day 4.
The average parasitemia was calculated as. The percentage suppression of parasitemia PSP for each plant extract was calculated as [ 21 ]. The chemoprotective prophylaxis in vivo study was set up and carried out by first treating the animals for four days with the different doses of the studied extracts before exposing them to infection. For each extract, 6 animals were selected as positive controls and negative controls and for test animals.
The survival of the mice in all groups was recorded twice a day. Parasitized erythrocytes were counted in Giemsa stained thin films prepared from tail blood on day 4. The percentage suppression of parasitemia for each plant extract was calculated. One-way analysis of variance ANOVA followed by Tukey's honest significant difference post hoc test was used to determine statistical significance in the comparisons of parasitemia suppression.
The results from the toxicity experiment showed that all animals in the ethyl acetate, methanol, and water extract groups were normal during the observations and at the end of the study period.
The mice of the isobutanol extract treatment group showed signs of acute toxicity. They showed signs of tremor on the third day. On a closer observation, the white fur seemed thin and slight and was erected. None of the animals experienced salivation, lacrimation, diarrhea, or convulsions.
Peters' 4-day suppressive test. Chemoprotective assay. Despite the overall favorable development in global malaria incidence and mortality rates, malaria still remains one of the gravest public health threats to human life in many regions.
Furthermore, the consequences of nonfatal malaria episodes pose a major economic burden to working age populations and local communities, especially in many African countries [ 5 ].
The current cost-effective options available in the prophylaxis and treatment of malaria are, regardless of their merits, widely considered insufficient to control malaria more efficiently. There is a need for development of new agents owing to the increasing resistance of the parasite to available agents [ 17 ]. We have recently shown antimicrobial activity of Croton macrostachyus H. Euphorbiaceae stem bark extracts against several human pathogenic bacteria and a fungus [ 19 ].
In the present study different crude stem bark extracts from C. The activity of C. These mostly amphiphile compounds have been described to protect erythrocytes against hypotonic hemolysis [ 24 ]. The main components of C. Because of their solubility properties one could conclude that the isobutanol and methanol extracts contained mixtures of these compounds. According to our previous work lupeol is extracted from C. Lupeol is a pharmacologically active triterpenoid with several potential medicinal properties.
There was a correlation between changes of the erythrocyte membrane shape to stomatocytic form and the inhibition of Plasmodium falciparum growth caused by a tropical plant Rinorea ilicifolia Kuntze lupeol in vitro [ 25 ]. In addition to indirect activity against P. In the present prophylaxis assay the impact of the most active ethyl acetate extract containing lupeol is linear and thus may reflect the indirect effect of lupeol on the erythrocyte membrane.
Activity of lupeol from the leaf hexane extract of Vernonia brasiliana L. Druce Compositae against P. In our study, two-, four-, and tenfold higher doses of lupeol in the ethyl acetate extract predominant compound of The activity implied chemoprotective, indirect activity on the erythrocyte membranes and was not found in the Peters' suppressive test supporting the results by De Almeida Alves et al.
However, because of the possible effect of other compounds or synergistic effect of several compounds present in the extract the role of lupeol against P. In the present study the in vivo assays showed that the studied extracts of C.
The most potential antimalarial chemotherapeutic and chemoprotective agent of the studied extracts was the ethyl acetate extract. Thus it can be considered as potential antimalarial drug only in low doses, although all the mice survived in the suppressive and chemoprotective assays.
Although the dose-response curves were not very steep, the extracts displayed suppression of parasitemia in a dose-dependent manner.
The exceptions were the isobutanol extract in both antimalarial tests and the ethyl acetate extract in the Peters' suppressive test. The effect of isobutanol extract may thus be due to nonspecific activity. These results are in parallel with the previous results by Ziegler et al.
In the chemoprotective assay the impact of the ethyl acetate extract was linear and may imply the indirect role of lupeol. The results obtained in the present study revealed that C. This upholds folkloric use of the plant and the earlier studies carried out with leaves, root, and fruit.
The ethyl acetate extract was the most promising candidate for further drug development. However, the active compounds of the extracts have not been identified, and the antimalarial activity of C. Further testing of the active components of C. They acknowledge the Kenya Medical Research Institute, Department of Malaria, for providing the opportunity to carry out the bioassays.
This study was funded by the Academy of Finland, Grant no. Consent to use the experimental animals in the study was obtained from the ethical committee of the Kenya Medical Research Institute, Center of Biotechnology and Research Development, Department of Malaria, Nairobi, Kenya.
Croton macrostachyus Hochst. Throughout its distribution area a decoction, infusion or maceration of leaves, stem bark or root bark are taken as a purgative and vermifuge. Leaf sap is used similarly. The seed oil is a very powerful purgative. In West Africa different plant parts in decoction are taken to treat constipation, stomach-ache and female infertility, and are used externally to treat stitch-like pain in the side and Guinea worm sores. In Ethiopia Croton macrostachyus has many uses. A leaf extract is applied against itchy scalp.
Burrows, J. Includes a picture. Page Da Silva, M. A preliminary checklist of the vascular plants of Mozambique. Dowsett-Lemaire, F. The flora and phytogeography of the evergreen forests of Malawi.
Croton macrostachyus is widely used as herbal medicine by the indigenous people of tropical Africa. The potential of C. The extensive literature survey revealed that C. The species is used as herbal medicine for diseases and ailments such as abdominal pains, cancer, gastrointestinal disorders, malaria, pneumonia, sexually transmitted infections, skin infections, typhoid, and wounds and as ethnoveterinary medicine.