The Antiplasmodial Agents of the Stem Bark of Entandrophragma Angolense (meliaceae)

In the search of active principles from the stem bark of Entandrophragma angolense, we submitted the compounds isolated from the dichloromethane – methanol (1:1) extract of the stem bark to antimalarial test against chloroquine resistant strain W2 of Plasmodium falciparum malaria parasite. Only 7α-obacunyl acetate and a cycloartane derivative exhibited a good activity, with IC 50 s of 2 and 5.4 µg/ml respectively. Other compounds were moderately active.


Introduction
Malaria is one of the most prevalent infections in the world.Despite more than a century of efforts to eradicate or control malaria, the disease remains a major and growing threat to the public health and economic development of countries in the tropical and subtropical regions of the world.In the search for new, safe and effective antimalarial drugs, screening of extracts from plants used in traditional medicine was indicated.
The plant family Meliaceae has been the subject of study as one of the most promising source of compounds with antiplasmodial properties.Some species belonging to this family (Azadirachta indica, Entandrophragma angolense, Entandrophragma candollei, Entandrophragma utile, Khaya grandifoliola) are widely used as antimalarials or antipyretics in traditional medicine (Irvine, 1961;Obih et al., 1986;Bray et al., 1990;Weenen et al., 1990a).
During the investigation of the plant species, Entandrophragma angolense Welwitsch C. D. C. (Meliaceae) we realised that the dichloromethane -methanol (1:1) extract of the stem bark of that plant is highly potent, inhibiting the development of the chloroquine resistant strain W2 of Plasmodium falciparum malaria parasite with an IC 50 of 18.8 µg/ml.This prompted us to go further and isolate compounds from that extract, in order to submit them to antiplasmodial activity against chloroquine resistant strain W2 of Plasmodium falciparum malaria parasite.

Plant materials
Stem bark of E. angolense Welwitsch C. D. C. was collected in April 1999 from the Awae forest reserve (Cameroon).The plant was identified, and a Voucher specimen (N° 29933) was deposited, by Dr. Achoundong of National Herbarium, Yaoundé (Cameroon).
From the chemotaxonomic point of view, it is of interest to note that, although the occurence of 7αacetoxydihydronomilin 1 and 7α-obacunyl acetate 2 had been reported in Uncaria genus (Rubiaceae) (Ahmed et al., 1978) and Citrus genus (Rutaceae) (Bennett and Hasegawa, 1982), respectively, this is the first time their isolation is reported from E. angolense.
In vitro antimalarial assay Cultivation of P. falciparum P. 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% O 2 , 5% CO 2 and 91% N 2 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.
Compounds were prepared as 20 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 controls contained 1 µM chloroquine phosphate (Sigma).Cultures were incubated at 37°C for 48 hrs (1 parasite erythrocytic life cycle).Parasites at ring stage were thereafter fixed by replacing the serum medium by an equal volume of 1% formaldehyde in PBS.Aliquots (50 µl) of each culture were then added to 5 ml round-bottom polystyrene tubes containing 0.5 ml 0.1% Triton X-100 and 1 nM YOYO nuclear dye (Molecular Probes) in PBS.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 (IC 50 s) calculated using Prism 3.0 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).
Different dilutions of the compounds were incubated at 37 o C with cultured W2 strain P. falciparum parasites for 48 hours.Parasites were thereafter fixed and stained, and parasitemias of treated and control cultures were determined.Results are means, compared to untreated controls, from 3 experiments.Error bars represent standard deviations of results.

Results
The phytochemical investigation of the stem bark of E. angolense (Meliaceae) led to the isolation of known limonoids 7α-acetoxydihydronomilin 1, 7α-obacunylacetate 2, methylangolensate 3, together with  The antiplasmodial activity of the isolated compounds have been evaluated against Plasmodium falciparum W2 strain.The IC 50 value of chloroquine for the clone W2 was 133 nM.The IC 50 values of the isolated compounds are shown in Table 1.From the results obtained, 7α-obacunyl acetate 2 and 24methylencycloartenol 6 were found to be significantly active in vitro against P. falciparum parasites, with IC 50 s of 2 and 5.4 µg/ml respectively.7α-acetoxydihydronomilin 1 and methylangolensate 3 showed moderate activity while tricosanoic acid 4 and 22-hydroxyhopan-3-one 5 were devoid of activity.

Discussion
Our work on the dichloromethane-methanol extract of the stem bark of E. angolense has resulted in the isolation of ten known compounds: 7α-acetoxydihydronomilin 1 (Ahmed et al., 1978) Six of the isolated compounds were submitted to antiplasmodial testing against Plasmodium falciparum malaria parasite, among which: 3 limonoids (1, 2 and 3), a pentacyclic triterpene (4), a fatty acid (5) and a tetracyclic triterpene (6).From the results obtained (Table 1), compound 2 and compound (6) were found to be significantly active against P. falciparum parasites with IC 50 s of 2.0 µg/ml and 5.4 µg/ml respectively.
The antimalarial activities of the limonoids in the present study are comparable to those of other limonoids and purified compounds obtained from other plants family (Noster and Kraus, 1990;Koumaglo et al., 1990;Bray et al., 1990, Weenen et al., 1990b, Nkunya, 1991;Bickii et al., 2000), the highest activity being observed with the 7α-obacunylacetate (2).
These compounds are quite different in their structures, which may explain the variations observed in their activities.Since the limonoids belong to the tetranortriterpenoid meliacolide class (share the same basic chemical structure), compounds (1) and ( 2) belong to this meliacolide class with an opened ring A. The good activity exhibited by compound (2) can be explained by the fact that (2) possesses an α, β-unsaturated carbonyl moiety at C1/C2 in ring A which was hypothesized to be involved in the Micheal addition reaction with the parasite nucleic acids (Weenen et al., 1990b).In fact, the reduction of the double bond in compounds possessing an α, β-unsaturated carbonyl moiety at C1/C2 in ring A has been shown to decrease the antimalarial activity (Bray et al., 1990).This observation agrees with the report that α, β-unsaturated ketone function is an important feature for enhanced antimalarial activity of quassinoids and terpenes which share similar chemical structures with limonoids (O' Neill et al., 1986;Weenen et al., 1990b;Phillipson and Wright, 1991).The difference of activity between compound (3) and the other two limonoids could be explained by the fact that compound (3) is a meliacolide with an opened ring B which probably decreases its activity.
The cycloartane derivative, 24-methylenecycloartenol was active against P. falciparum parasites with an IC 50 of 5.4 µg/ml.Although its chemical structure is to be well-confirmed, the difference of activity observed between this compound and compound 2 might be explained by the difference in their chemical structures.So, compound 2 is a tetracyclic triterpene with a furanic lateral chain and a lactonized ring A, while compound (6), although being also a tetracyclic triterpene, possesses a non cyclic lateral chain.It is of interest to notice that, the presence of the cyclopropyl ring in the structure of compound ( 6) should be taken into account in the explanation of its activity.Further work based on the study of the structure-activity relationship will permit to elucidate that phenomenon.

Conclusion
Limonoids isolated from the stem bark of E. angolense have been shown to exhibit moderate in vitro antimalarial activities against P. falciparum parasites.Two compounds: 7α-obacunylacetate and 24methylenecycloartenol were the most active.The results of this study can be correlated to the traditional use of E. angolense in the treatment of malaria.Further studies are however necessary to evaluate the toxicity and to elucidate the mechanism of action of the active compounds.Combinations with other antimalarial drugs may be interesting in the goal of fighting drug resistance.

Table 1 :
IC 50 values of isolated compounds against Plasmodium falciparum strain W2.The results are expressed as geometric means ± SD from triplicate separate experiments.