Antimalarial activity of some plants traditionally used in treatment of malaria in Kwale district of Kenya

Journal of Ethnopharmacology 112 (2007) 545–551

Antimalarial activity of some plants traditionally used in treatment of malaria in Kwale district of Kenya C.N. Muthaura a,∗ , G.M. Rukunga a , S.C. Chhabra b , S.A. Omar c , A.N. Guantai d , J.W. Gathirwa a , F.M. Tolo a , P.G. Mwitari a , L.K. Keter a , P.G. Kirira a , C.W. Kimani a , G.M. Mungai e , E.N.M. Njagi f a

Centre for Traditional Medicine and Drug Research, Kenya Medical Research Institute, P.O. Box 54840, 00200 Nairobi, Kenya b Department of Chemistry, Kenyatta University, P.O. Box 43844, 00100 Nairobi, Kenya c Centre for Biotechnology Research and Development, Kenya Medical Research Institute, P.O. Box 54840, 00200 Nairobi, Kenya d University of Nairobi, School of Pharmacy, P.O. Box 30197, 00100 Nairobi, Kenya e East Africa Herbarium, National Museums of Kenya, P.O. Box 40658, 00100 Nairobi, Kenya f Department of Biochemistry and Biotechnology, Kenyatta University, P.O. Box 43844, 00100 Nairobi, Kenya Received 20 September 2006; received in revised form 29 March 2007; accepted 24 April 2007 Available online 5 May 2007

Abstract Methanolic and water extracts of five medicinal plant species used for treatment of malaria in traditional/cultural health systems of Kwale people in Kenya were tested for antimalarial activity against Plasmodium falciparum and Plasmodium berghei, respectively and for their cytotoxic effects. The most active extracts (IC50 < 10 ␮g/ml) screened against chloroquine (CQ) sensitive (D6) and resistant (W2) P. falciparum clones, were the water and methanol extracts of Maytenus undata (Thunb.) Blakelock (Celasteraceae), methanol extracts of Flueggea virosa (Willd.) Voigt (Euphorbiaceae), Maytenus putterlickioides (Loes.) Excell and Mendoca (Celastraceae), and Warburgia stuhlmannii Engl. (Canellaceae). These extracts showed various cytotoxic levels on Vero E6 cells with the water extract of M. undata exhibiting least cytotoxicity. At least one of the extracts of the plant species exhibited a high chemo suppression of parasitaemia >70% in a murine model of P. berghei infected mice. These results indicate that there is potential for isolation of a lead compound from the extracts of the five plants. W. stuhlmannii and M. putterlickioides have not been reported before for antiplasmodial activity. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Antiplasmodial activity; Warburgia stuhlmannii; Maytenus putterlickioides

1. Introduction Malaria is a major tropical parasitic disease responsible for significant morbidity and mortality and in the absence of practical preventive measures; the current options are chemoprophylaxis and chemotherapy. A dramatic recrudescence of malaria is ongoing due to the increasing resistance of vectors to insecticides and the progressive resistance of the parasite, mainly Plasmodium falciparum to drugs. The increasing prevalence of strains of P. falciparum resistance to chloroquine (CQ) which had been efficacious, safe, accessible and affordable poses

∗

Corresponding author at: Kenya Medical Research Institute, P.O. Box 70174, 00400 Nairobi, Kenya. Tel.: +254 20 2722541; fax: +254 20 2720030. E-mail address: [email protected] (C.N. Muthaura). 0378-8741/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2007.04.018

a serious problem for malaria control (Trape, 2002) and leaves Africa with unprecedented situation in which the only affordable treatment options are rapidly losing therapeutic efficacy (Fidock et al., 2004). Drug resistance strains of P. falciparum have been found in many endemic areas of the world and majority of conventional antimalarial drugs have been associated with treatment failure (Olliaro and Bloland, 2001). These developments and the difficulty of creating efficient vaccines coupled with adverse side effects of the existing antimalarial drugs underline the urgent need for novel, well tolerated and more efficient antimalarial drugs (Bickii et al., 2000), affordable to the poor, living in malaria endemic tropical countries. In endemic countries, accessible treatments against malaria are mainly based on the use of traditional herbal remedies. Indeed, indigenous plants play an important role in the treatment of many diseases (Phillipson and Wright, 1991) and 80%

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of the people worldwide are estimated to use herbal remedies (Geoffrey and Kirby, 1996; Phillipson, 1994). However, few data are available on their efficiency and safety, despite the fact that validation of traditional practices could lead to innovative strategies in malaria control. Natural products represent a virtually inexhaustible reservoir of molecules, most of which are hardly explored and could constitute lead molecules for new antimalarial drugs, such as artemisinin, isolated from Artemisia annua (Kayser et al., 2003). Several studies have been undertaken to evaluate the inhibitory effects of various plants extracts on P. falciparum (Le Tran et al., 2003; Wanyoike et al., 2004) in culture. The in vivo antimalarial properties of several plant extracts have been studied on Plasmodium berghei (Andrade-Neto et al., 2003), P. vinckei (Moretti et al., 1994) and P. yoelii infected mice (Brandao et al., 1992). Following this trend, this study presents the results obtained from the evaluation of the in vivo antimalarial and in vitro antiplasmodial activities of five plants used in Kenyan folk medicine against malaria in Kwale district: Flueggea virosa (Willd.) Voigt (Euphorbiaceae) CM 118, Warburgia stuhlmannii Engl. (Canellaceae) CM 119, Harungana madagascariensis Poir (Guttiferae) CM 128, Maytenus putterlickioides (Loes.) Excell and Mendoca (Celastraceae) CM 116 and Maytenus undata (Thunb.) Blakelock (Celasteraceae) CM 133. The possible cytotoxic activities of these plants were determined using Vero E6 cells (Kurokawa et al., 2001) and acute toxicity was determined as described in the General guidelines for methodologies on research and evaluation of traditional medicine (WHO/EDM/TRM/2000.1). 2. Materials and methods

filtered and then freeze dried. Methanol extracts (50 g powder in 500 ml of solvent) were prepared by maceration of the plant material with MeOH at room temperature for 48 h. The mixture was filtered and the filterate concentrated to dryness in vacuo. The yields of the water extracts were higher than the corresponding methanol extracts and ranged between 4.5 and 12.8%, while those of the methanol extracts were between 1.6 and 8.4%. The dry solid extracts were stored at −20 ◦ C in airtight containers until used. 2.3. Parasites To test for antimalarial activities of the aqueous and methanol extracts, the mouse-infective, CQ sensitive P. berghei strain ANKA donated by International Livestock Research Institute, ILRI, Kenya was used. The CQ susceptible (D6) and resistant (W2) clones donated by the Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Washington DC were used to test the antiplasmodial activities of the plant extracts in vitro. 2.4. Preparation of test extracts Stock solutions of aqueous extracts (500 ␮g/ml) were made in distilled deionized water and filter sterilized using 0.22 ␮m membrane filters in a laminar flow hood. The methanol extracts were dissolved in DMSO (Sigma chemical CO., St. Louis, MO, USA) followed by subsequent dilution to lower concentration of DMSO, to <1% to avoid carry over (solvent) effect (Dorin et al., 2001). Reference drugs, CQ diphosphate and Artemisinin at a concentration of 1 ␮g/ml each, were similarly prepared and all solutions stored at −20 ◦ C until used.

2.1. Plant materials 2.5. Cultures of Plasmodium falciparum The five plant samples used in this study were collected in December 2004 from Kwale district of Kenya based on ethno pharmacological use through interviews with local communities and traditional health practitioners (THP). Information gathered included vernacular names (in parentheses) and the parts used in preparation of the herbal antimalarial remedies: F. virosa (Mukwamba) leaves, M. undata (Muriakitu) leaves, M. putterlickioides (Muthuthi) root bark, H. madagascariensis (Mukokotsaka) leaves and W. stuhlmannii (Mkaa) stem bark. The plants were identified by a taxonomist at the East Africa Herbarium, National Museums of Kenya, Nairobi where voucher specimens were deposited. The plant parts were chopped into small pieces; air dried at room temperature (25 ◦ C) under shade and pulverized using a laboratory mill (Christy & Norris Ltd., England). 2.2. Preparation of extracts Considering that people in Kwale usually use hot water to prepare their herbal remedies as decoctions and sometimes concoctions, aqueous hot infusions of each plant part were prepared (50 g of powdered material in 500 ml of distilled water) in a water bath at 60 ◦ C for 1 h. The extracts that were obtained were

P. falciparum culture of D6 (CQ sensitive isolate from Sierra Leorne) and W2 (CQ resistant isolate from Indochina) were used in the study. The culture media was a variation of that described by Trager and Jensen (1976) and consisted of RPMI 1640 supplemented with 10% serum (Schlichtherle et al., 2000). Uninfected human blood group O+ erythrocytes (<28 days old) served as host cells. The cultures were incubated at 37 ◦ C in an atmosphere of 3% CO2 , 5% O2 , and 92% N2 (BOC® , Nairobi). 2.6. Bioassay The in vitro semi automated microdilution assay technique that measures the ability of the extracts to inhibit the incorporation of [G-3 H]hypoxanthine (Amersham International, Burkinghamshire, UK) into the malaria parasite was used (Desjardins et al., 1979; Muregi et al., 2003). For the test, 25 ␮l aliquots of culture medium were added to all the wells of a 96 well flat-bottom microculture plate (Costar Glass Works, Cambridge, UK). Aliquots (25 ␮l) of the test solutions were added, in triplicate, to the first wells, and a Titertek motorized hand diluter (Flow Laboratories, Uxbridge, UK) was used to make two-fold serial dilutions of each sample over a 64-fold

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concentration range. The susceptibility tests were carried out with intial parasitaemia (expressed as the percentage of erythrocytes infected) of 0.4% by applying 200 ␮l, 1.5% haematocrit, P. falciparum culture to each well. 200 ␮l of culture media without parasites was added into four wells on the last row of each plate to serve as the background. Parasitized and non-parasitized erythrocytes were incubated at 37 ◦ C in a gas mixture 3% CO2 , 5% O2 and 92% N2 . After 48 h each well was pulsed with 25 ␮l of culture medium containing 0.5 ␮Ci of [G-3 H]hypoxanthine and the plates were incubated for a further 18 h. The contents of each well were then harvested onto glass fiber filter mats using a 96 well harvester, washed thoroughly with distilled water and dried. [G-3 H]hypoxanthine uptake was determined using a micro beta trilux liquid scintillation and luminescence counter (Wallac Micro Beta Trilux). Computation of the concentration of drug causing 50% inhibition of [G-3 H] hypoxanthine uptake (IC50 ) was carried out by interpolation after logarithmic transformation of both concentration and cpm values using the formula: IC50 =

antilog(log X1 + [(log Y50 − log Y1 )(log X2 − log X1 )] (log Y2 − log Y1 )

where Y50 was the cpm value midway between parasitisized and non-parasitisized control cultures and X1 , Y1 , X2 , and Y2 were the concentrations and cpm values for the data points above and below the cpm midpoints (Sixsmith et al., 1984).

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extract using the formula: (A − B) A × 100 where A was the mean parasitaemia in the negative control and B was the parasitaemia in the test group (Tona et al., 2001). Extract activity was determined by %reduction of parasitaemia in treated groups compared with untreated infected mice. For all the groups of experimental mice used, survival time in days was recorded and the mean for each group calculated. 2.8. Acute toxicity Healthy Swiss female mice weighing 20–22 g were divided into groups of five in each cage and had access to tap water and food, except for a short fasting period (12 h) before oral administration of a single dose of the extract. The water extracts were dissolved/suspended in distilled water and administered by gavage at logarithmic dose ranging between 500 and 5000 mg/kg body weight to give five dose levels of 500, 889.15, 1581.18, 2811.80 and 5000 mg/kg body weight. The general behaviour of mice was observed continuously for 1 h after the treatment and then intermittently for 4 h, and thereafter over a period of 24 h (Twaij et al., 1983). The mice were further observed for up to 14 days following treatment for any signs of toxicity, and the latency of death. The LD50 value was determined according to a method described by Thompson (1985). 2.9. Cell cytotoxicity assay

2.7. In vivo determination of antimalarial activity Chloroquine sensitive P. berghei strain ANKA was used to assess the in vivo intrinsic antimalarial activity. The assay protocol was based on 4-day suppressive test (Peters et al., 1975). The parasite strain was maintained by serial passage of blood from an infected mouse to a naive mouse. Female Swiss mice (6–7 weeks old; 20–22 g) were randomly infected by intraperitoneal (i.p.) inoculation of 107 erythrocytes parasitized with P. berghei in a saline suspension of 0.2 ml on day zero (D0) and allocated to several groups of five mice in each cage. They were fed on standard pellets and water ad libitum. The animals were housed in the Animal House at KEMRI (Kenya Medical Research Institute) and the Institute’s Animal Care and Use Committee gave approval for the study. Plant extracts were solubilized in 10% tween 80 (methanol extracts) or in saline (water extracts) and administered once daily intraperitoneally (D0 to D3) at a concentration of 100 mg/kg/day in a dose volume of 0.2 ml. Two groups (five mice each) served as negative and positive controls, respectively. The negative group received saline/tween 80 while the positive group was treated with reference drug CQ diphospate at a dose of 5 mg/kg/day i.p. Each day from D1 to D4 thin blood smears were made from the tail of each mouse, stained with 10% Giemsa in phosphate buffer, pH 7.2 and examined microscopically for assessment of parasitaemia. The mean parasitaemia in each group of mice on day 4 was used to calculate the %chemo suppression for each

The cytotoxic concentration causing 50% cell lysis and death (CC50 ) was determined for the extracts by a method described by Kurokawa et al. (2001). Vero cells, an established cell line from kidney cells of African green monkey (Vero E6) were seeded at a concentration of 2.5 × 104 cells/well in a 24 well plates and grown in minimum essential medium (MEM) at 37 ◦ C under 5% CO2 for 48 h. The culture media (MEM) was replaced by fresh media containing extract at various concentrations, and the cells further grown for 24 h. The cells were treated with trypsin and the number of viable cells determined by the tryphan blue exclusion method. The concentration of herbal extract reducing cell viability by 50%, CC50 was determined from a curve relating percent cell viability to the concentration of the extract. 2.10. Statistical analysis The Welch’s t-test was used to test the significance of differences between mean results obtained for different samples, and Dunnett’s test was used for multiple comparisons of significance between results of the same sample means against controls (Graph Pad InstatTM V2.04). Values with p < 0.05 were considered statistically significant. 3. Results and discussion The in vitro activities (IC50 ) of the extracts against CQ sensitive (D6) and resistant (W2) P. falciparum clones and

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Table 1 In vitro antiplasmodial activity (IC50 ± S.D.) of the extracts of selected medicinal plants against P. falciparum D6 and W2 clones and cytotoxicity with selectivity index on mammalian Vero E 6 cells Plant species

Extract

F. virosa

MeOH Water

2.28 ± 0.17 25.52 ± 2.60b,c

M. undata

MeOH Water

7.40 ± 1.40c 0.95 ± 0.06b

M. putterlikioides

MeOH Water

4.41 ± 0.16c 204.67 ± 0.20b,c

10.26 ± 0.30f,a >250

112.4 ± 4.5 380.8 ± 6.7

25.5 nd

H. madagascariensis

MeOH Water

39.07 ± 1.57c 105.0 ± 0.04b,c

43.70 ± 1.45f,a >250

461.5 ± 18.7 3569.2 ± 45.1

11.8 nd

W. stuhlmannii

MeOH Water

2.33 ± 0.16 >250 31.32 ± 2.15 3.38 ± 0.49

233.0 ± 16.5 3337.8 ± 15.9

128.7 nd

Chloroquine (ng/ml) Artemisinin (ng/ml)

P. falciparum (D6) (␮g/ml)

1.81 152.86 8.97 0.90

± ± ± ±

0.04 12.6b,c 1.18 0

P. falciparum (W2) (␮g/ml)

Vero E6 cells CC50 (␮g/ml)

Selectivity index CC50 /D6

3.64 ± 0.91a 37.80 ± 2.23d,e,a

682.6 ± 18.3 2990.5 ± 51.2

299.3 117.1

9.89 ± 0.84a 1.90 ± 0.03d,e

251.3 ± 18.3 3645.7 ± 32.8

33.9 3836.8

The IC50 values are expressed as mean ± S.D. of three different determinations per experiment; SI: selectivity index, is defined as the ratio of the CC50 value determined on the mammalian cell line on the IC50 value determined on P. falciparum (D6); Two reference drugs, chloroquine and artemisinin are included as positive controls and their IC50 are given in ng/ml. nd: not determined (IC50 , D6 > 100 ␮g/ml). a p < 0.05, MeOH/water W2 vs. CQ. b p < 0.05, water D6 vs. MeOH D6. c p < 0.05 (MeOH/water D6 vs. CQ). d p < 0.05, water W2 vs. water D6. e p < 0.05, water W2 vs. MeOH W2. f p < 0.05, MeOH W2 vs. MeOH D6.

cytotoxicities (CC50 ) on mammalian Vero E6 cells are summarized in Table 1. Selectivity index (SI) of each extract is also presented in Table 1. SI was defined as the ratio of the CC50 value on the mammalian cells to the IC50 value on P. falciparum. Extracts with a high selectivity for the parasites offer the potential for safer therapy. The activity was categorized as high when IC50 was <10 ␮g/ml, moderate between 10 and 20 ␮g/ml and weak between 20 and 100 ␮g/ml. Extracts having activity beyond this range were considered inactive. The two clones were highly susceptible (IC50 < 5 ␮g/ml) to the methanol extracts of F. virosa and W. stuhlmannii as well as the water extract of M. undata and their ratio of sensitivity for the two clones was less than that of chloroquine suggestive of no cross resistance with the latter. The distribution of antiplasmodial constituents was apparently more in methanol extracts than in water extracts. The water and methanol extracts of M. undata were the most active with both extracts displaying high activity (IC50 < 10 ␮g/ml) for both clones. The CC50 for the methanol extract (251.3 ␮g/ml) was higher than that of the water extract (3645.7 ␮g/ml), which was the lowest in the plants studied, but both were much lower than their corresponding IC50s indicative of high selectivity for the malaria parasites. Indeed, all cytotoxicity values of the extracts on Vero cells were lower than corresponding IC50s on P. falciparum. This was consistent with in vivo safety of the water extract, where no acute toxicity was observed in mice (LD50 > 5000 mg/kg.). The water extract, although more active than the methanol extract, showed lower chemo suppression of parasitaemia in P. berghei infected mice (Table 2). The results for in vivo antimalarial activity do not necessarily correlate with those for in vitro antimalarial activity as

reported by Gessler et al. (1995). This may be due to biotransformation of the constituents or poor bioavailability of the active compounds in vivo. However, both extracts had similar survival time (13.0 and 15.0 days) as the reference drug CQ (p > 0.05). In South Africa, Clarkson et al. (2004) reported antiplasmodial activity of M. undata leaves. Several 12-oleanene triterpene and secotriterpene acids have been isolated from aerial parts of M. undata (Muhammad et al., 2000). The methanol extract of F. virosa was highly active, while the water extract was in the weak activity category. The CC50 for the methanol and water extract were weak (SI, 299.3 and 117.1, respectively) and similarly there was no acute toxicity in mice (LD50 > 5000 mg/kg). The methanol extract was twice (chemo suppression 70.91%) as active as its water extract (35.15%), however the survival time for P. berghei infected mice treated with water extract was significantly longer (13.0 days) than that of the methanol extract (11.0 days). It is probable that some extracts that prolonged survival time despite mild chemo suppressive activity or those extracts with high chemo suppressive activity but had less significant survival time may have been due to mechanisms of drug action such as having an indirect effect on the immune system, or by other pathways that are not yet understood (Rasoanaivo et al., 1992). On the other hand, the suppressed development of P. berghei infection in the first days of infection suggests that the extract affects the blood stages of the parasites. The subsequent development of infection and shorter survival time points to a shorter duration of action of the extract, perhaps limited by rapid metabolism or elimination. It is surprising that Kraft et al. (2003) reported no activity for F. virosa, while other studies in South Africa (Clarkson et al., 2004) reported good activity consistent with our results. The

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Table 2 Mean (x ± S.D.) parasite density, chemo suppression and survival time of P. berghei infected mice treated intraperitonially with methanol and aqueous extracts at a dose of 100 mg/kg body weight, once a day for 4 days Plant species

Extract

Mean parasite density (pd)a

%Chemo suppression

F. virosa

MeOH Water

8.91 ± 1.32b 19.87 ± 2.21d,e

70.91 ± 4.53 35.15 ± 1.13

11.0 ± 1.0c 13.0 ± 1.0f

M. undata

MeOH Water

7.27 ± 0.37b 16.02 ± 2.04d,e

76.29 ± 9.01 47.74 ± 4.24

13.0 ± 1.0f 15.0 ± 2.65f

M. putterlickioides

MeOH Water

6.54 ± 0.28b 16.76 ± 3.97d,e

78.66 ± 5.71 45.31 ± 5.23

13.33 ± 1.53f 14.67 ± 2.08f

H. madagascariensis

MeOH Water

14.37 ± 1.13b 3.67 ± 1.13e

53.13 ± 5.46 88.04 ± 3.57

11.0 ± 1.0c 11.33 ± 1.53c

W. stuhlmannii

MeOH Water

14.42 ± 1.73b 6.55 ± 1.90d,e

52.95 ± 4.31 84.95 ± 6.00

11.33 ± 1.53c 12.33 ± 1.53f,c

30.65 ± 1.62 0.03 ± 00

0.0 ± 0 99.89 ± 0.13

8.0 ± 1.0 16.33 ± 1.53

Saline/10% tween 80 Chloroquine

Mean survival time (days)

The results are expressed as mean ± S.D. of five determinations per experiment. a Parasite densities (pd) in percentages. (p < 0.05), MeOH and water extracts vs. controls. b p < 0.05, MeOH extracts vs. chloroquine. c p < 0.05, mean survival time of mice treated with extracts vs. chloroquine. d p < 0.05, water extracts vs. chloroquine. e p < 0.05, MeOH vs. water extracts. f p < 0.05, mean survival time of mice treated with extracts vs. negative controls.

potency of the extract may depend on solvent of extraction, georeference, time and season of harvesting or other environmental factors (Prance, 1994). Kraft et al. (2003) reports that due to dry season when plants were collected, it was not possible in some cases to collect the traditionally used plant parts. The plant is traditionally used for malaria in Tanzania and South Africa (Hedberg et al., 1983; Watt and Breyer-Brandwijk, 1962). The roots and fruits are chewed for treatment of snakebite and a cold extract of the roots is drunk for stomachache (Kokwaro, 1993). Root decoction is also used for chest pains (Beentje, 1994). The roots are reported to contain the alkaloids norsecurinine, dihyronorsecurinine and hordenine (Hedberg et al., 1983). Novel C,C-linked dimeric indolizidine alkaloids, flueggenines A (1) and B (2), as well as their biosynthetic precursor norsecurinine have recently been isolated from the plant (Gan et al., 2006). The presence of bergenin, an isocoumarin in substantial amounts in the methanol leaves extract has been reported to be antiprotozoal (Nyasse et al., 2004). The methanol extract of M. putterlickioides exhibited moderate in vitro activity and high chemo suppression (78.66%). Although the water extract was inactive (IC50 > 100 ␮g/ml), it showed moderate chemo suppression (45.31%). The survival time for the two extracts was similar to that of CQ (p > 0.05). Gessler et al. (1995) found high in vitro (IC50 , 0.62 ␮g/ml) and in vivo activities (chemo suppression 89.9%) for M. senengalensis EtOAc root bark extract with no overt signs of toxicity in mice, but the extract was cytotoxic when tested on HT and KB cell lines. These activities were consistent with our findings for the two related species; M. undata and M. putterlickioides MeOH extracts whose SI (33.9 and 25.5) were relatively low compared to other extracts. The root extracts have been shown to inhibit protein kinase C activity, as well as lethality against brine shrimp. Dihydroagarofuran sesquiter-

pene alkaloids mayteine, putterine A and putterine B have been isolated from the root extract (Schaneberg et al., 2001). Quinonemethide triterpenoids are another diverse group of secondary metabolites from a related species Maytenus ilicifolia (Buffa Filho et al., 2002), which have revealed potential anti-tumour and anti-microbial activities (Bavovada et al., 1990). The various biological activities have been attributed to those diverse secondary metabolites among them maytansinoids (Reider and Roland, 1984). Maytenus putterlickioides leaf is used in East Africa for hookworm infestations and the root bark as an aphrodisiac and as a remedy for laziness (Kokwaro, 1993). The methanol extract of H. madagascariensis showed weak in vitro activity and moderate chemo suppression (53.13%). The water extract was inactive in vitro but exhibited high chemo suppression of parasitaemia (88.04%). Gessler et al. (1995) reported H. madagascariensis stem bark petroleum ether extract had in vitro activity of IC50 , 10 ␮g/ml and a chemo suppression of 29.3% on P. berghei infected mice. He also reported a low SI for the root bark extract on KB cell line, consistent with our results. In East Africa a decoction of the stem bark of H. madagascariensis is drunk as a remedy for malaria. Root and stem bark infusion is used to hasten development of breasts in young women and to interrupt menses (Kokwaro, 1993). Isolation of anthraquinones, saponins, and steroids from the plant has been reported (Tona et al., 1998). Antibacterial flavanone, astilbin has also been reported (Moulari et al., 2006). Representatives of these classes of compounds isolated from other plant sources have been reported to inhibit P. falciparum growth in vitro and/or in vivo (Nkunya et al., 1991; Bickii et al., 2000; Nundkumar and Ojewole, 2002) and are likely to be responsible for the observed activities. The methanol extract of W. stuhlmannii was highly active but the water extract was not active. However, the latter exhibited

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high chemo suppression of parasitaemia (84.95%) in P. berghei infected mice and the survival time was significant compared to negative control. The methanol extract exhibited weak cytotoxicity (SI, 128.7) while the water extract was not toxic to mice at a concentration of 5000 mg/kg. Flavonols and sesquiterpenes have been isolated from the leaves and stem bark of W. stuhlmannii (Manguro et al., 2003). Several other naturally occurring sesquiterpenes have been found with promising schizonticidal activity (Thebtaranonth et al., 1995). The activity of W. stuhlmannii could presumably be ascribed to the presence of the sesquiterpenes in addition to flavonols, which collectively may be involved in the antiplasmodial activity. Methanol extracts were generally more active in vitro than water extracts probably due to active lipophilic constituents, which do not extract into the water extract. The traditional method of preparation often involves boiling in water for quite some time and it is not uncommon to make a concoction of more than one plant part, which would possibly enhance the extraction of lipophilic constituents in the aqueous phase and act in synergy against the malaria parasites in humans. The water extracts of M. undata and F. virosa, were active in vitro; the other water extracts though inactive exhibited relatively high chemo suppression in P. berghei infected mice. These were probably prodrugs metabolized into active constituents in vivo. This may explain the consistent reports by traditional healers that these plants are effective in treating malaria in humans. Both activities as well as the safety studies further confirm their ethnopharmacological usefulness as antimalarials. The plant extracts with IC50 < 10 ␮g/ml offer potential for isolation of lead antimalarial compounds or characterization of some active compounds that could be used as markers for standardization of the extracts for use as traditional antimalarials, and in this way contribute to the development of potential antimalarial medicine from the Kenyan ethnobotany. Acknowledgements This work received financial support from UNICEF/ UNDP/World Bank/WHO special programme for Research and Training in Tropical Diseases (TDR). We thank the Director, KEMRI for allowing publication of this study. References Andrade-Neto, V.F., Brandao, M.G., Stehmann, J.R., Oliveira, L.A., Krettli, A.U., 2003. Antimalarial activity of Cinchona-like plants used to treat fever and malaria in Brazil. J. Ethnopharmacol. 87, 253–256. Bavovada, R., Blasko, G., Shien, H.-L., Pezzuto, J.M., Cordell, G.A., 1990. Spectral assignment and cytotoxicity of 22-hydroxytingenone from Glyptopetalum sclerocarpum. Planta Med. 56, 380–382. Beentje, H.J., 1994. Kenya Trees, Shrubs and Lianas. National Museums of Kenya, Nairobi, Kenya. Bickii, J., Njifulie, N., Foyere, J.A., Basco, L.K., Ringwald, P., 2000. In vitro antimalarial activity of limonoids from Khaya grandifoliola C.D.C. (Meliaceae). J. Ethnopharmacol. 69, 27–33. Brandao, M.G.L., Grandi, T.S.M., Rocha, E.M.M., Sawyer, D.R., Krettli, A.U., 1992. Survey of medicinal plants used as antimalarials in the Amazon. J. Ethnopharmacol. 36, 175–182.

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