Introduction:

Malaria a preventable and curable disease causes over 1 million deaths mostly in children below the age of 5 years and 350500 million episodes of acute illness each year WHO 2006. This disease affects more than 50 of the Worlds population and hits tropical Africa hardest. This region bears more than 90 of the global malaria burden Breman et al. 2004 WHO 2006.rnrnMalaria morbidity and mortality have been held in check by the widespread availability of cheap and effective antimalarial drugs. Resistance of Plasmodium falciparum to these drugs may represent the single most important threat to the health of people in tropical countries. Chloroquine CQ had been the mainstay of antimalarial drug treatment for over 40 years but resistance became widespread Bloland et al. 1993 WHO 2006. In Kenya chloroquine CQ and sulphadoxine-pyrimethamine SP had been the drugs of choice. Both these antimalarials costed less than US 0.20 per adult treatment course but the drugs required to treat drug resistant falciparum malaria quinine mefloquine and halofantrine were over ten times more expensive and could not be afforded by most countries in the tropics especially those in Africa. Resistance to chloroquine and SP led to the use of artemisinin based antimalarial drugs Ronn et al. 1996 WHO 2003. In Kenya the first line drug for uncomplicated malaria is artemether-lumefantrine Coartem Ministry of Health 2006. Reports of reduced efficacy of artemisinins against P. falciparum from Southeast Asia is worrying Dondorp et al. 2010 Ferrer-Rodiriguez et al. 2004 Noedl et al. 2009.rnrnMalaria remains a leading cause of morbidity and mortality in Kenya especially in young children and pregnant women. Each year there are over 8.2 million malaria infections. It accounts for 30 of outpatient attendances and 19 of admissions to health facilities Jean-Marie 2002 Ministry of Health 2006. The disease is endemic in the lowlands particularly the coastal strip and Lake Victoria basin where transmission is sufficiently intense. Both incidence and prevalence of infection reach more than 90 of the population within 1012 weeks after the beginning of the rainy season Hoffman et al. 1996 Dianne et al. 2003 Ministry of Health 2006.rnrnWe have previously reported the antimalarial activity of other plants from Kenya Gathirwa et al. 2008 Gathirwa et al. in press Irungu et al. 2007 Kigondu et al. 2009 Kirira et al. 2006 Muregi et al. 2003 Muthaura et al. 2007 Rukunga et al. 2008. We have also isolated some promising antimalarial compounds from plants Kirira et al. 2007 Mutai et al. 2004 Rukunga and Waterman 1996. In continuation of our search for antimalarial compounds/extract from folkrole plants the antimalarial and cytotoxicity activities of some medicinal plant extracts previously documented by Koch et al. 2005 used by the Maasai community in the traditional medicine in Kenya were determined. These included Sericocomopsis hildebrandtii Schinz Pentas lanceolata Forssk. Defleurs Fuerstia africana T.C.E. Friers Manilkara discolor Sond. J.H.Hemsl. and Acacia tortilis Hayne.

Objectives:

Assessing antiplasmodial and cytotoxicity activities of some selected plants used by the Maasai community Kenya

Methodology:

2.1. Collection of plant materialsrnThe plant materials were collected in Kajiado District and identified by Mr. Patrick Kyalo Mutiso a plant taxonomist at the University of Nairobi. The plant material leaves seeds roots and stem were dried and pulverized.rnrn2.2. Extraction and fractionationrn500 g amount of the powder were extracted sequentially with solvents of increasing polarity hexane or petroleum ether dichloromethane or chloroform and lastly methanol. The extraction was done at room temperature with intermittent shaking for 48 h. After filtration the solutions were evaporated completely at reduced pressure and the afforded crude extracts were further dried under a stream of nitrogen for 24 h and used for the biological assays. The most active extract was further fractionated using Vacuum Liquid Chromatography VLC. Various combinations of solvents were used at different ratios and similar fractions were pooled after running Thin Layer Chromatography TLC.rnrn2.3. Bioassayrn2.3.1. In vitro determination of antiplasmodial activityrnThe plant extracts were assayed using an automated micro-dilution technique to determine 50 growth inhibition of cultured parasites Chulay et al. 1983 Desjardins et al. 1979. Two different strains of P. falciparum were used in this study the chloroquine sensitive Sierra Leone D6 and chloroquine resistant Indochina W2. They were grown in a continuous culture supplemented with mixed gas 92 nitrogen 5 carbon dioxide and 3 oxygen 10 human serum and 6 hematocrit of O red blood cells. Once cultures reached the optimum growth growth rate 3.0 and a parasitaemia of 3 with at least 80 ring developmental stage present parasite solution was transferred to a 96 well micro-titer plate with wells pre-coated with the test sample. The test sample was serially diluted across the plate to provide a range of concentrations used to determine IC50 values. Plates were incubated in a gas chamber for 48 h after which 3H-hypoxanthine was added and parasites allowed to grow for a maximum of 18 h. Cells were processed with a 96 well plate harvester MicroBeta onto filtermat paper Wallac and washed to eliminate unincorporated isotope. Filters were measured for radio-activity in a micro-titer plate scintillation counter Wallac. Data from the counter was processed using Oracle database program to determine IC50 values.rnrn2.3.2. Cytotoxicity assay procedurernThe method described by Mosmann 1983 was used. Briefly vero cells were grown to a confluent monolayer in a 75 cm2 culture flask at 37 C in humidified 5 CO2 in minimum essential medium MEM with Eagles salt containing 2 L-glutamine supplemented with 10 FBS Fetal Bovine Serum. The cells were trypsinized and 1.0 ml aliquots of medium containing approximately 2.5 104 cells/ml seeded into 24-well culture plates and incubated for 2 days at 37 C humidified 5 CO2. The medium was removed and replaced with fresh medium containing test sample at various concentrations 100 200 and 400 g/ml prepared in MEM and 10 bovine serum in triplicate. For each test sample three wells were used as control non-drug-treated cells. After a further 24 h incubation the medium was aspirated from the wells and cells removed from monolayer culture by incubating with trypsin-EDTA 250 L for 5 min at 37 C. 50 L of the cell suspension was then transferred into a 96 well plate. Trypan blue 50 L was then added to the cell suspension in each well. The number of viable cells was determined by counting the cells with the aid of a haemocytometer and percentage viability determined using the standard procedure. The concentration of the test sample reducing cell viability by 50 CC50 value of the control was estimated from a curve relating percent cell viability to the concentration of sample.

Findings:

Results:

plant species and the parts of the plants which were collected extracted and tested biologically. On the other hand Table 2 shows the amounts of raw material used for extraction against the percentage yields of the extracts. The IC50 values and the selectivity indices SI of the tested successive plant extracts are presented in Table 3. Activity of extracts is considered high if IC50 10 moderate between 11 and 50 mild between 51 and 100 g/ml and inactive 100 g/ml Muregi et al. 2003. The degree of selectivity an extract shows for the malaria parasite can be expressed by its selectivity index SI value:rnthe tested crude plant extracts 3 crude extracts showed high antiplasmodial activity against D6 strain of P. falciparum with IC50 values lower or equal to 10 g/ml 4 were moderately active with IC50 values ranging between 10 and 50 g/ml. The aerial parts and roots of F. africana demonstrated interesting antiplasmodial activity against the chloroquine sensitive plasmodial strain D6 IC50 1.56 0.00 and 4.6 0.57 g/ml respectively with a selectivity index of 44 against vero cells. These results were also observed for chloroquine resistant W2 plasmodium strain IC50 2.5 0.4 and 6.3 0.1 g/ml respectively. Ethyl acetate extract of the roots of P. lanceolata and the aerial parts of S. hildebrandtii had moderate antiplasmodial activity against D6 and W2 IC50 14.3 1.6 and 16.51 0.91 g/ml respectively. The petroleum ether extract of the aerial parts of P. lanceolata exhibited moderate activity against the W2 IC50 33.91 1.06 g/ml. M. discolor also exhibited promising antiplasmodial activity especially against D6 IC50 11.5 3.2 and 26.6 4.4 g/ml for methanol extracts of the leaves and ethyl acetate extract of the stem bark respectively. Extracts of P. lanceolata and S. hildebrandtii showed weak cytotoxicity Table 3.rnrnBased on the high antiplasmodial activity and selectivity index values obtained for the petroleum ether extract of the aerial parts of F. africana fractionation was carried out using VLC and antiplasmodial activity and cytotoxicity of the fractions was determined and compared with that of original extract

Conclusion:

The results obtained in this study generally show that the plant F. africana has high antiplasmodial activity. Though the IC50 values for the cytotoxicity assay are quite low for the plant selectivity index 1 is an indication of minimal cytotoxicity. Specifically the aerial parts of F. africana exhibited strong antiplasmodial activity against the chloroquine sensitive strain D6 IC50 1.56 0.00 and 4.6 0.57 g/ml respectively with a selectivity index of 44 and 24 against vero cells and against the chloroquine resistant W2 strain IC50 2.5 0.4 and 6.3 0.1 g/ml respectively with a selectivity index of 27 and 4 against vero cells. This is well in agreement with literature values where the leaves of F. africana have been reported to exhibit high in vitro antiplasmodial activity IC50 3.8 g/ml CHCl3 extract and has displayed selectivity for the malaria parasite P. falciparum as indicated by a lack of cytotoxicity ED50 20 g/ml against cultured KB cells Koch et al. 2005.rnrnInterestingly after carrying out fractionation of the petroleum ether extract of the aerial parts of F. africana antiplasmodial activity of the various VLC fractions was lowered from IC50 1.56 to values between 3.03 and 14.20 g/ml against D6. This is an indication that the compounds in the extract may be acting synergistically hence leading to high antiplasmodial activity of the total petroleum ether extract.rnrnEthyl acetate extract of the roots of P. lanceolata and the aerial parts of S. hildebrandtii exhibited moderate antiplasmodial activity against D6 and W2 IC50 14.3 1.6 and 16.51 0.91 g/ml respectively. The petroleum ether extract of the aerial parts of P. lanceolata was also showing moderate activity against the W2 IC50 33.91 1.06 g/ml. However the roots of P. lanceolata and S. hildebrandtii have been reported to exhibit high in vitro antiplasmodial activity IC50 5.2 and 3.8 g/ml CHCl3 extract and have also displayed selectivity for the malaria parasite P. falciparum as indicated by a low cytotoxicity ED50 20 g/ml against cultured KB cells Koch et al. 2005. The difference between the literature values and the results obtained may be due to the fact that the plants in the current study and those of literature were collected during different seasons of the year. This leads to differences in chemical profiles of the extracts from the plants hence a difference in biological activities. No literature information has been found for M. discolor which in the current study was exhibiting some promising antiplasmodial activity.rnrnPrevious chemical investigation on the leaves of P. lanceolata revealed the presence of asperuloside an iridoid monoterpene and a series of iridoid glucosides Inouye et al. 1988 Schripsema et al. 2007. An antimalarial abietane diterpene has been isolated from F. africana Koch et al. 2006. Isolation of active compounds from the various plants in this study has not been exhaustively achieved according to literature and hence they should be pursued further.

Author(s):

Focus County(s):

Kajiado County

Programme Area(s):

Natural Products Research & Drug Development (NAPREDA)

Research Priority Area(s):

Disease Domain(s):

Malaria treatment

Publication Date: 03.Aug.2011

Conference Title:

Venue: