Journal of Ethnopharmacology 123 (2009) 483–488
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Antiplasmodial activity of extracts from seven medicinal plants used in malaria treatment in Cameroon Fabrice Fekam Boyom a,∗ , Eugénie Madiesse Kemgne a,b , Roselyne Tepongning a , Vincent Ngouana a , Wilfred Fon Mbacham b , Etienne Tsamo c , Paul Henri Amvam Zollo a , Jiri Gut d , Philip J. Rosenthal d a
Laboratory of Phytobiochemistry and Medicinal Plants Study, Faculty of Science, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon Laboratory for Public Health Biotechnology, Biotechnology Center, University of Yaoundé I, Cameroon Department of Organic Chemistry, Faculty of Science, TWAS Research Unit, University of Yaoundé I, Cameroon d Division of Infectious Diseases, Department of Medicine, University of California San Francisco, CA 94943, USA b c
a r t i c l e
i n f o
Article history: Received 31 December 2008 Received in revised form 19 February 2009 Accepted 5 March 2009 Available online 20 March 2009 Keywords: Malaria Uvariopsis congolana Polyalthia oliveri Enantia chlorantha Artocarpus communis Dorstenia convexa Croton zambesicus Neoboutonia glabrescens Acetogenin Antiplasmodial
a b s t r a c t Aim of the study: In a search for new plant-derived biologically active compounds against malaria parasites, we have carried out an ethnopharmacological study to evaluate the susceptibility of cultured Plasmodium falciparum to extracts and fractions from seven Cameroonian medicinal plants used in malaria treatment. We have also explored the inhibition of the Plasmodium falciparum cysteine protease Falcipain-2. Materials and methods: Plant materials were extracted by maceration in organic solvents, and subsequently partitioned or fractionated to afford test fractions. The susceptibility of erythrocytes and the W2 strain of Plasmodium falciparum to plant extracts was evaluated in culture. In addition, the ability of annonaceous extracts to inhibit recombinant cysteine protease Falcipain-2 was also assessed. Results and discussion: The extracts showed no toxicity against erythrocytes. The majority of plant extracts were highly active against Plasmodium falciparum in vitro, with IC50 values lower than 5 g/ml. Annonaceous extracts (acetogenin-rich fractions and interface precipitates) exhibited the highest potency. Some of these extracts exhibited modest inhibition of Falcipain-2. Conclusion: These results support continued investigation of components of traditional medicines as potential new antimalarial agents. © 2009 Published by Elsevier Ireland Ltd.
1. Introduction The heavy toll exacted by malaria is compounded by increasing drug resistance. In Cameroon, where drug resistance is widespread, malaria is the leading cause of morbidity and mortality. The emergence and spread of resistance to chloroquine and sulfadoxine–pyrimethamine have led to recommendations that they be replaced with artemisinin-based combination therapies (ACTs), which offer much improved efficacy. However, the development of resistance to artemisinins or their partner drugs may severely limit the utility of ACTs. Therefore, research to develop alternative therapies is greatly needed. To this end, plants used in traditional medicines may offer a promising source of compounds with antimalarial activity. Indeed, natural products and their derivatives have traditionally been a common source of drugs, and represent more than 30% of the current pharmaceutical market (Kirkpatrick, 2002; Newman et al., 2003; Njomnang Soh and Benoit-Vical, 2007).
∗ Corresponding author. Tel.: +237 7727 6585. E-mail address:
[email protected] (F.F. Boyom). 0378-8741/$ – see front matter © 2009 Published by Elsevier Ireland Ltd. doi:10.1016/j.jep.2009.03.008
According to an ethnobotanical survey in Cameroon (Adjanohoun et al., 1996), some 137 species from 48 different families are used in traditional medicine alone or in mixtures to treat malaria and/or fever. These plants represent more than half of the Cameroonian medicinal species used in such instances (Titanji et al., 2008). Up to date, there is no evidence in the literature that many of these plants have been scientifically investigated to establish whether or not they have antimalarial activity. In this paper, we report the results of an ethnopharmacological study in which, in vitro susceptibility of red blood cells and reference Plasmodium falciparum resistant strain (W2) to plant extracts and fractions was evaluated. In addition, given that proteases that hydrolyze hemoglobin to provide amino acids for parasite protein synthesis are among the potential targets for drugs directed against Plasmodium falciparum, the inhibition of cysteine protease falcipain-2 by annonaceous extracts was also assessed. The extracts were obtained from seven Cameroonian medicinal plants belonging to three families, Annonaceae, Moraceae, and Euphorbiaceae, which are locally used in traditional medicine to cure many diseases including malaria and fevers. Ethnobotanical data were collected by personal contact and interviews with traditional healers in the Nfoundi division of the Centre Province of Cameroon. Criteria of
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literature and chemotaxonomical surveys afterward guided the selection of the seven species. 2. Materials and methods 2.1. Plant collection The plant samples were collected in Mt. Eloundem in Yaoundé neighbourhood in March 2007. The plant species were identified by Mr. Victor Nana, who is ethnobotanist at the National Herbarium of Cameroon where voucher specimens have been deposited under the following identification numbers: Uvariopsis congolana (De Wild) Fries: 37016/HNC Polyalthia oliveri Engl.: 19416 SRF/Cam Enantia chlorantha Oliv.: 32065/SRF/Cam Artocarpus communis J.R. & G. Forst: 43982 HNC Dorstenia convexa De Wild: 53450 HNC Croton zambesicus Muell. Arg.: 8204/SRFCam Neoboutonia glabrescens Müll. Arg. Prain: 7433/SRFCam
the cycles), and centrifuged at 12,000 × g for 30 min at 4 ◦ C. The pellet was washed twice with 2.5 M urea, 20 mM Tris–Cl, 2.5% Triton X-100, pH 8.0; centrifuged at 17,000 × g for 30 min at 4 ◦ C; and solubilized in 6 M guanidine HCl, 20 mM Tris–Cl, 250 mM NaCl, 20 mM imidazole, pH 8.0 (5 ml/g of inclusion body pellet) at RT for 60 min with gentle stirring. Insoluble material was separated by centrifuging at 27,000 × g for 30 min at 4 ◦ C. For purification of the recombinant protein, the supernatant was incubated for 60 min at RT with a nickel–nitrilotriacetic acid (Ni–NTA) resin (Qiagen) equilibrated with the same buffer. The resin was loaded on a column and washed with 10 bed volumes each of 6 M guanidine HCl, 20 mM Tris–Cl, 250 mM NaCl, pH 8.0; 8 M urea, 20 mM Tris–Cl, 500 mM NaCl, pH 8.0; and 8 M urea, 20 mM Tris–Cl, 30 mM imidazole, pH 8.0. Bound protein was eluted with 8 M urea, 20 mM Tris–Cl, 1 M imidazole, pH 8.0, and quantified by the Bradford dye binding assay (Bradford, 1976). The Ni–NTA-purified recombinant falcipain-2 was reduced with 10 mM DTT at 37 ◦ C for 45 min and refolded to an active protease as described by Shenai et al. (2000). 2.4. Evaluation of the biological activities
2.2. Plant extraction and extracts partition Air-dried and ground plant materials (500 g) were extracted according to the following methods. For Annonaceae samples, the extracts were prepared following the method described by Alali et al. (1999) that was designed to prepare acetogenin-rich fractions, with little modifications. The materials were submitted to 95% ethanol extraction for 48 h to have the ethanolic residues. The ethanolic residues were partitioned between H2 O and CH2 Cl2 , leading to H2 O layers and CH2 Cl2 layers. The residues of the CH2 Cl2 layers were partitioned between 90% MeOH and hexane to give MeOH layers and hexane layers. The MeOH layers were submitted to vacuum evaporation to obtain acetogenin-rich fractions, viz. UcS, UcL, PoSb, EcSb, and EcS obtained from the respective plant organs. The interface precipitates were also obtained during residues partition between CH2 Cl2 /H2 O (EcSb1, EcS1) and MeOH/hexane (EcSb2, EcS2). All the afforded residues and interface precipitates were evaluated for biological activity. For the Moraceae and Euphorbiaceae samples, air-dried and ground plant parts (500 g) were macerated in methanol for 48 h. The filtrates were evaporated under reduce pressure and the residues obtained used for further experiments. The crude extracts were subjected to open column fractionation using hexane–ethyl acetate solvent systems of increasing polarity. Crude extracts and the afforded fractions were evaluated for biological activity. The yields given in Table 1 were calculated in percentage relative to dried material weight (w/w). 2.3. Expression and purification of recombinant falcipain-2 The carboxyl-terminal 35 amino acids fragment of falcipain-2 (–35FP2) was amplified from the pTOP–FP2 plasmid using Vent DNA polymerase (New England BioLabs) and primers as described previously (Shenai et al., 2000). The PCR product was digested with BamHI and HindIII, gel-purified, and ligated into the pQE-30 expression vector (which encodes an aminoterminal 6-His tag; Qiagen) to produce expression construct pQ–35FP2. This construct was used to transform M15 (pREP4)-strain Escherichia coli, its sequence was confirmed, and transformants were analyzed for expression of the protein by SDS-PAGE. Bacteria containing pQ-35FP2 were grown to mid-log phase and induced with isopropyl-1-thio-ˇd-galactopyranoside (IPTG, 0.25 mM) for 5 h at 37 ◦ C. Cells were harvested, washed with ice-cold 100 mM Tris–Cl, 10 mM EDTA, pH 7.4, sonicated (12 cycles of 10 s each, with cooling for 10 s between
2.4.1. Evaluation of erythrocyte susceptibility to plant extracts A preliminary toxicological assessment was carried out to determine the highest drug concentrations that can be incubated with erythrocytes without any significant damage. This was done according to the 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide/phenazine methosulfate (MTT/PMS) colorimetric assay described by Cedillo-Rivera et al. (1992), with some modifications. This method is based on the reduction of MTT to formazan by enzymes of viable cells. The extracts were serially diluted in 96 well culture plates, and each concentration incubated in triplicate with erythrocytes (2% hematocrit) in a final 100 L culture volume (at 37 ◦ C, in a 3% O2 , 5% CO2 and 91% N2 atmosphere, in the presence of RPMI 1640, 25 mM HEPES, pH 7.4 for 48 h). At the end of the incubation period, the cultures were transferred into polypropylene microcentrifuge tubes and centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. 1.5 ml MTT solution with 250 g PMS were added to the pellets. Controls contained no erythrocytes. The tubes were thereafter incubated for 45 min at 37 ◦ C, and then centrifuged, and the supernatant was discarded. The pellets were re-suspended in 0.75 ml of HCl 0.04 M in isopropanol to extract and dissolve the dye (formazan) from the cells. After 5 min, the tubes were vigorously mixed and centrifuged, and the absorbance of the supernatant was determined at 570 nm. The tubes containing the most viable erythrocytes produced more formazan (highest O.D.). From the results obtained, the highest drug concentrations producing minimal damage to the cells were considered as starting points for further drug dilutions. 2.4.2. Evaluation of the antiplasmodial activity Plasmodium falciparum strain W2, which is resistant to chloroquine and other antimalarials (Singh and Rosenthal, 2001), was cultured in sealed flasks at 37 ◦ C, in a 3% O2 , 5% CO2 and 91% N2 atmosphere in RPMI 1640, 25 mM HEPES, pH 7.4, supplemented with heat inactivated 10% human serum and human erythrocytes to achieve a 2% hematocrit. Parasites were synchronized in the ring stage by serial treatment with 5% sorbitol (Sigma) (Lambros and Vanderberg, 1979) and studied at 1% parasitemia. Plant extracts were prepared as 1 mg/ml stock solutions in DMSO, diluted as needed for individual experiments, and tested in triplicate. The stock solutions were diluted in supplemented RPMI 1640 medium so as to have at most 0.2% DMSO in the final reaction medium. An equal volume of 1% parasitemia, 4% hematocrit culture was thereafter added and gently mixed thoroughly. Negative controls contained equal concentrations of DMSO. Positive
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Table 1 Yields of plant extraction/partition; in vitro susceptibility of Plasmodium falciparum to extracts and inhibition of recombinant falcipain-2. Plant family/species Annonaceae
Crude extracts Acetogenin-rich
Uvariopsis congolana (stem) Uvariopsis congolana (leaf) Polyalthia oliveri (stem bark) Enantia chlorantha (stem bark) Enantia chlorantha (stem bark) Enantia chlorantha (stem bark) Enantia chlorantha (stem) Enantia chlorantha (stem) Enantia chlorantha (stem)
UcS UcL PoSb EcSb
e
Fractions/precipitates
3.5 3.2 3.9 2.9 1.2 0.8 4.6 0.6 0.4
EcSb1 EcSb2 EcS EcS1 EcS2
Plant family/species Moraceae
Crude extracts Extract
Artocarpus communis (stem bark)
MeOH (ACsb)
Artocarpus communis (Leaf)
MeOH (ACle)
Dorstenia convexa (Twigs)
MeOH (DCtg)
Plant family/species Euphorbiaceae
Crude extracts Extract
Croton zambesicus (Stem bark)
MeOH (Czsb)
N. glabracens (leaf) N. glabracens (stem bark)
MeOH (Neole) MeOH (Neosb)
Yield
e
Fractions/precipitates Fraction
Yield
f1
IC50 ± S.D. (g/ml)
4.47 4.57 4.30 2.06 0.68 0.82 4.79 0.85 14.72 f1
± ± ± ± ± ± ± ± ±
0.45 0.76 0.31 0.01 0.08 0.16 1.09 0.34 8.87
IC50 ± S.D. (g/ml)
100% hex (ACsb1) Hex:EtOAc 75:25 (ACsb2) Hex:EtOAc 50:50 (ACsb3) Hex:EtOAc 25:75 (ACsb4) 100% EtOAc (ACsb5)
8.20 2.20 1.30 0.87 0.94 0.67
>50 1.69 ± 0.05 >50 2.34 ± 0.04 >50 >50
100% Hex (ACle1) Hex:EtOAc 75:25 (ACle2) Hex:EtOAc 50:50 (ACle3) Hex:EtOAc 25:75 (ACle4) 100% EtOAc (ACle5)
8.32 3.25 1.47 1.31 0.44 0.24
4.00 ± 0.37 3.58 ± 1.00 2.26 ± 0.58 10.81 ± 2.54 4.36 ± 0.60 >50
100% Hex (DCtg1) Hex:EtOAc 75:25 (DCtg2) Hex:EtOAc 50:50 (DCtg3) Hex:EtOAc 25:75 (DCtg4) 100% EtOAc (DCtg5)
8.95 3.32 2.56 0.41 0.35 0.28
>50 >50 >50 4.38 ± 1.41 3.71 ± 0.89 3.08 ± 0.99
e
Fractions/precipitates Fraction 100% hex (Czsb1) Hex:EtOAc 75:25 (Czsb2) Hex:EtOAc 50:50 (Czsb3) Hex:EtOAc 25:75 (Czsb4) 100% EtOAC (Czsb5)
Yield
f1
IC50 ± S.D. (g/ml)
9.14 3.45 1.95 1.12 0.88 0.92
5.69 ± 0.06 >50 >50 >50 6.26 ± 1.05 >50
7.56 8.22
5.50 ± 0.20 >50
f2
IC50 ± S.D. (g/ml)
>50 >50 >50 22.71 ± 1.88 18.15 ± 0.70 >50 >50 38.85 ± 1.42 30.08 ± 0.54 f2
IC50 ± S.D. (g/ml)
f2
IC50 ± S.D. (g/ml)
Positive controls CQ E-64
0.03 ± 0.01 0.028 ± 0.002
e
The percent extraction yields were calculated in w/w. The susceptibility of erythrocytes and the W2 strain of Plasmodium falciparum to plant extracts was evaluated; the ability of annonaceous extracts to inhibit recombinant cysteine protease falcipain-2 was also assessed; f1/f2 Concentration that killed/inhibited 50% of parasites/enzyme relative to negative control. S.D. = standard deviation, the drugs were tested in triplicate. Positive controls were CQ = chloroquine and E-64 = l-Transepoxy-succinyl-leucylamido-(4guanidino)-butane.
controls contained 1 M chloroquine phosphate (Sigma). Cultures were incubated at 37 ◦ C for 48 h (one parasite erythrocytic life cycle). Parasites at the ring stage were thereafter fixed by replacing the serum medium by an equal volume of 1% formaldehyde in PBS. Aliquots (50 l) of each culture were then added to 5 ml round-bottom polystyrene tubes containing 0.5 ml 0.1% Triton X100 and 1 nM YOYO nuclear dye (Molecular Probes) in PBS, and parasitemias of treated and control cultures were compared using a Becton-Dickinson FACSort flow cytometer to count nucleated (parasitized) erythrocytes. Data acquisition was performed using CellQuest software. These data were normalized to percent control activity and 50% inhibitory concentrations (IC50 ) were calculated using Prism 4.0 software (GraphPad) with data fitted by non linear regression to the variable slope sigmoidal dose–response formula, y = 100/1 + 10(logIC50 −x)H where H is the hill coefficient or slope factor (Singh and Rosenthal, 2001).
2.4.3. Falcipain-2 inhibition assay Activity was measured as hydrolysis of the fluorogenic substrate benzyloxycarbonyl-Leu-Arg-7-amino-4-methyl-coumarin (Z-LeuArg-AMC) by using microplate reactions and a Fluoroskan II spectrofluorometer, as described previously (Rosenthal et al., 1996). For inhibitor assays, recombinant falcipain-2 prepared as described previously (Shenai et al., 2000) was incubated with inhibitors (added from stocks concentrated 100- to 1000-fold in dimethyl sulfoxide [DMSO]) with 10 mM dithiothreitol, pH 5.5, for 30 min at room temperature before the substrate (50 M) was added. Equal concentrations of enzyme were used for each experiment. Multiple inhibitor concentrations were studied in triplicate, and the rates of hydrolysis were determined and compared to those of controls containing equal concentrations of DMSO. All values were normalized to the control activity, and 50% inhibitory concentrations (IC50 ) were calculated with the Prism 4.0 program
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(GraphPad Software), with data being fitted by nonlinear regression as above. 3. Results 3.1. Plant extraction The yields of plant extraction and partition were calculated relatively to the weight of the starting dried materials (500 g) and presented in Table 1. 3.2. The biological activities Test extracts showed toxicity to erythrocytes at concentrations above 0.5 mg/ml, many orders of magnitude above concentrations with antiplasmodial activity. The great majority of the tested extracts showed antiplasmodial activity in vitro against the W2 strain of Plasmodium falciparum. All the relevant results obtained are summarized in Table 1. First, all the acetogenin-rich fractions and interface precipitates from Annonaceae species showed high potency. In almost all cases, the IC50 values were found to be lower than 5 g/ml, the most active being the interface precipitates (EcSb1 , EcSb2 ) from the stem bark and EcS1 from the stem of Enantia chlorantha (0.68 g/ml, 0.82 g/ml, and 0.85 g/ml, respectively). The acetogenin-rich fractions also exhibited very interesting potency with IC50 values ranging from 2.06 g/ml to 4.79 g/ml. The only exception to this rule was the interface precipitate EcS2 , from the stem of Enantia chlorantha that exerted moderate activity with an IC50 value of 14.72 g/ml. In addition, 4/9 annonaceous extracts (EcSb, EcSb1, EcS1, and EcS2) were found to inhibit the recombinant cysteine protease falcipain-2 with IC50 values ranging from 18.15 g/ml to 38.85 g/ml, EcSb1 being the most active. The other residues obtained from these plants showed no activity. Second, almost all fractions from the three Moraceae samples (Artocarpus communis-stem bark and leaf, Dorstenia convewa-twigs) showed high potency against W2 Plasmodium falciparum in vitro with IC50 values below 5 g/ml. In this case, the only crude extract that exhibited such potency is ACle, as well as its subsequent fractions (ACle1-2, 4). Third, the Euphorbiaceae extracts also showed antiplasmodial activity, mainly the crude from the stem bark of Croton zambesicus (Czsb) with an IC50 value of 5.69 g/ml and the one from Neoboutonia glabrescens (IC50 = 5.5 g/ml). 4. Discussion and conclusions The activity levels showed by extracts from the Annonaceae species may be partly attributed to acetogenins. Indeed, the annonaceous acetogenins are the most powerful of the known inhibitors of complex I (NADH: ubiquinone oxidoreductase) in mitochondrial electron transport systems (Lewis et al., 1993); in addition, they are potent inhibitors of NADH oxidase of plasma membranes (Morré et al., 1995); these enzymes are all found in Plasmodium falciparum. These actions decrease oxidative, as well as cytosolic ATP production. The consequence of such ATP deprivation is a programmed cell death. Moreover, they are potent cytotoxics with insecticidal, ascaricidal, fungicidal, antiparasitic, bactericidal and antiplasmodial activities (Rupprecht et al., 1990; Alali et al., ˜ et al., 2000; Rakotomanga et al., 2004). It has been 1999; Guadano shown that acetogenins inhibit cancer cells that are multidrug resistant (Oberlies et al., 1995, 1997a,b). Thus, they thwart biological resistance, by interacting with lipid bi-layers, where they exert their activity on membrane-bound enzymes (Alali et al., 1999). Considering these activities on some vital plasmodial enzymes, we have hypothesized that acetogenins may be efficient inhibitors
of proteases as well. To assess this hypothesis, all the annonaceous extracts viz. UcS, UcL, PoSb, EcSb, EcS, EcSb1, EcSb2, EcS1, and EcS2 were evaluated against the recombinant cysteine protease falcipain-2. From the results achieved, there is supporting evidence that these compounds might be contributing in the overall extracts activity. Further studies (purification of individual acetogenins, assessment of their inhibitory activity) will enable us to definitely conclude on this issue. The three Annonaceae species that have been investigated in this study, viz. Uvariopsis congolana, Polyalthia oliveri and Enantia chlorantha are used in malaria cure in Cameroon. Previous studies indicate that water and ethanolic extracts, as well as purified alkaloids from the stem bark of Enantia chlorantha showed interesting antiplasmodial activities in vitro and in vivo (Agbaje and Onabanjo, 1991, 1994; Agomo et al., 1992; Adjanohoun et al., 1996; Kimbi and Fagbenro-Beyioku, 1996; Vennerstrom and Klayman, 1998). The ethanolic extract from a related species, Enantia polycarpa from Côte d’Ivoire also showed good potency in vitro against strain K1 of Plasmodium falciparum (Kamanzi Atindehou et al., 2004). Apart from a study carried out by Quevauviller and Hamonniere (1977) on the activity of the principal alkaloids of Polyalthia oliveri on the central nervous and cardiovascular systems, very few data is available on this species. Related species such as Polyalthia cerasoides; Polyalthia viridis; Polyalthia evecta have been extensively investigated for biological activities (Ichino et al., 2006; Kanokmedhakul et al., 2006, 2007). Uvariopsis is an African specific genus that has been reported to produce acetogenins. To the best of our knowledge, no study has yet been reported on Uvariopsis congolana, but related species have already been investigated. Of note, extracts from Uvariopsis congensis reported to be eaten by wild chimpanzees (Pan troglodytes schweinfurthii) in the Kibale National Park, western Uganda, were assessed for potential chemotherapeutic values. The authors found that active compounds isolated from some plants occasionally ingested by chimpanzees, including Diospyros abyssinica (Ebenaceae), Uvariopsis congensis (Annonaceae), and Trichilia rubescens (Meliaceae) showed highly significant medicinal properties as antibacterial, antimalarial, and/or antileishmania (Krief et al., 2006). The two Moraceae, Artocarpus communis and Dorstenia convexa are locally used for the management, control and treatment of various types of human diseases, including fevers and malaria. Dorstenia convexa is an herbaceous plant that grows widely in the mountain grasslands of central Africa and Artocarpus communis is a tree of moderate size, the only species of the genus Artocarpus found in Cameroon. Several biological activities have been reported for the stem bark, leaves and roots of these plants. An aqueous infusion of the whole Dorstenia convexa plant is a typical preparation employed as anti-inflammatory (Wei et al., 2005), hyperglycaemic (Adewole and Ojewole, 2007), and against ear oedema (Koshihara et al., 1988). Artocarpus communis has also antiplatelet effect (Weng et al., 2006), antinephritis and radical scavenging activity (Fukai et al., 2003). The genera Artocarpus and Dorstenia are rich sources of phenolic metabolites such as prenyl flavonoids from the heartwood of Artocarpus communis with inhibitory activity on lipopolysaccharide-induced nitric oxide production (Han et al., 2006) and geranyl flavonoids that exhibited moderate cytotoxicity against some human cancer cells (Wang et al., 2007). Omisore et al. (2005) reported the antitrichomonal and antioxidant activities of the leaves and twigs extracts and compounds of Dorstenia convexa. Although numerous bioactive agents have been isolated from the roots, stem bark and leaves of Artocarpus communis and Dorstenia convexa, few antimalarial studies have been reported to date. The investigation of a related species (Artocarpus integer) led to the isolation of prenylated stilbene with an IC50 of 1.7 g/ml against Plasmodium falciparum in culture (Boonlaksiri et al., 2000).
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The extracts of two Euphorbiaceae viz. Croton zambesicus and Neoboutonia glabrescens were also investigated in this study. These species are locally used to treat various affections, including malaria and fevers. Croton zambesicus is a diterpenes-rich species that has been extensively investigated and that showed various biological activities (Boyom et al., 2002; Baccelli et al., 2005, 2007). Recently, Okokon et al. (2005) have evaluated the antiplasmodial activity of the ethanolic leaf extract of this species. Their results showed considerable activity against Plasmodium berghei in mice. Concerning Neoboutonia glabrescens, no previous scientific investigation has been reported. The methanol and aqueous extracts of a closely related species, Neoboutonia macrocalyx growing in Kenya were tested for brine shrimp lethality and in vitro antiplasmodial activity against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum (NF54 and ENT30) (Kirira et al., 2006). The results achieved from this work are encouraging and lead to a twofold main conclusion. First, they support the use of the investigated species in traditional medicine for antimalarial therapy. Second, some of the extracts such as those from Annonaceae species might exert their antiplasmodial activity through the potency of acetogenins, a fatty acid-derived class of secondary metabolites which are mainly found in this plant family. This activity may be exerted through the inhibition of some vital parasitic enzymes such as cysteine proteases. Further detailed investigations will enable us to clarify this assertion. Acknowledgements This investigation was supported by a grant from the National Institutes of Health, USA (AI35707) to PJR, who is a Distinguished Clinical Scientist of the Doris Duke Charitable Foundation. We gratefully acknowledge the practical help of Mr. Victor Nana of the Cameroon National Herbarium for his assistance with the collection and identification of plant materials. References Adewole, S.O., Ojewole, J.O., 2007. Hyperglycaemic effect of Artocarpus communis Forst (Moraceae) root bark aqueous extract in Wistar rats. Cardiovascular Journal of Africa 18, 221–227. Adjanohoun, J.E., Aboubakar, N., Dramane, K., Ebot, M.E., Ekpere, J.A., Enoworock, E.G., Focho, D., Gbile, Z.O., Kamanyi, A., Kamsu, K.J., Keita, A., Mbenkum, T., Mbi, C.N., Mbiele, A.C., Mbome, J.C., Muberu, N.K., Nancy, W.L., Kongmeneck, B., Satabie, B., Sofowora, A., Tamze, V., Wirmum, C.K., 1996. Traditional Medicine and Pharmacopoeia: Contribution to ethnobotanical and floristic studies in Cameroon. Ed. Organization of African Unity; Scientific, Technical and Research Commission. Agbaje, E.O., Onabanjo, A.O., 1991. The effects of extracts of Enantia chlorantha in malaria. Annals of Tropical Medicine and Parasitology 85, 585–590. Agbaje, E.O., Onabanjo, A.O., 1994. Toxicological study of the extracts of anti-malarial medicinal plant Enantia chlorantha. Central African Journal of Medicine 40, 71–73. Agomo, P.U., Idigo, J.C., Afolabi, B.M., 1992. Antimalarial” medicinal plants and their impact on cell populations in various organs of mice. African Journal of Medicine and Medical Science 21, 39–46. Alali, F.Q., Liu, X.X., Mc Laughlin, J.L., 1999. Annonaceous acetogenins: recent progress. Journal of Natural Products 62, 504–540. Baccelli, C., Block, S., Van Holle, B., Schanck, A., Chapon, D., Tinant, B., Meervelt, L.V., Morel, N., Quetin-Leclercq, J., 2005. Diterpenes isolated from Croton zambesicus inhibit KCl-induced contraction. Planta Medica 71, 1036–1039. Baccelli, C., Navarro, I., Block, S., Abad, A., Morel, N., Quetin-Leclercq, J., 2007. Vasorelaxant activity of diterpenes from Croton zambesicus and synthetic trachylobanes and their structure–activity relationships. Journal of Natural Products 70, 910–917. Boonlaksiri, C., Oonanant, W., Kongsaeree, P., Kittakoop, P., Tanticharoen, M., Thebtaranonth, Y., 2000. An antimalarial stilbene from Artocarpus integer. Phytochemistry 54, 415–417. Boyom, F.F., Keumedjio, F., Dongmo, P.M.J., Ngadjui, T.B., Amvam Zollo, P.H., Menut, C., Bessiere, J.M., 2002. Essential oils from Croton zambesicus Muell. Arg. growing in Cameroon. Flavour and Fragrance Journal 17, 215–217. Bradford, M.M., 1976. Analytical Biochemistry 72, 248–254. Cedillo-Rivera, R., Ramfrez, A., Munoz, O., 1992. A rapid colorimetric assay with the Tetrazolium salt MTT and Phenazine Methosulfate (PMS) for viability of Entamoeba histolytica. Archives of Medical Research 23, 59–61. Fukai, T., Satoh, K., Nomura, T., Sakagami, H., 2003. Antinephritis and radical scavenging activity of prenylflavonoids. Fitoterapia 74, 720–724.
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