Antiplasmodial activity of Aspidosperma indole alkaloids.
The antiplasmodial activity of twelve alkaloids with an aspidospermane skeleton was estimated in vitro on chioroquine-resistant and sensitive strains of Plasmodium falciparum. Seven tetracyclic alkaloids possessing a free ethyl chain such aspidospermine, showed [IC.sub.50] after incubation for 72 h between 3.2 and 15.4 [micro]M. Moreover, four pentacyclic alkaloids with ethyl chain included in a tetrahydrofuran, such haplocine, showed a reduced activity, with [IC.sub.50], after 72 h, between 22.6 and 52.6 [micro]M. According to these results, a chloroquine-potentiating experiment was also performed with two of the most active compounds. Isoholograms were obtained and demonstrated a synergic effect of N-formyl-aspidospermidine and aspidospermine when associated with chloroquine. The cytotoxicity and the selectivity index of some alkaloids were also estimated.
Key words: Aspidosperma pyrifolium, A. megalocarpon, Aspidospermane alkaloids, antiplasmodial activity, Plasmodium falciparum
In many tropical and subtropical regions, malaria represents the major parasitic infection, with the most severe forms caused by Plasmodium falciparum. Faced with the increase in the resistance of P. falciparum to traditional treatments, several research groups have estimated the antiplasmodial activity of indolomonoter-penoid alkaloids to find new antimalarial compounds of plant origin. In this field, alkaloids isolated from species of Picralima (Iwu et al., 1994), Alstonia and Strychnos genera (Wright et al., 1992, 1994, Frederich et al., 1999), possessing monomeric, dimeric and quasidimeric skeletons were tested but no clear relationships between structure and activity were deduced. Moreover, during a phytochemical investigation of two Aspidosperma species, A. pyrifolium Mart. and A. megalocarpon Mull. Arg. (Apocynaceae), indolo-monoterpenoid alkaloids possessing the aspidospermane skeleton were isolated (Mitaine et al., 1996, 1998). In this series of compounds, eleven were chosen for their close structura l similarity and, for the first time, their in vitro antiplasmodial activity and toxicity were examined. As some alkaloids are known to have modulating effects on chloroquine activity (Frappier et al., 1996), we have tested two of the most active ones, N-formyl aspidospermidine 3 and aspidospermine 5, in association with chloroquine, in order to determine the existence of a possible cumulative effect between these molecules.
Materials and Methods
Two strains were cultured continuously in vitro according to Trager and Jensen, 1976: A chioroquine-sensitive strain of Plasmodium falciparum, from Nigeria, with an inhibitory concentration ([IC.sub.50]) for chloroquine at 79 nM and a chloroquine-resistant strain, FcM29-Cameroon, with [IC.sub.50] at 445 nM. The [IC.sub.50] values of chloroquine were checked every 2 months and no significant variations were observed (Mustofa et al., 2000).
The antiplasmodial activity of the alkaloids was evaluated according to a method derived from Desjardins et al., 1979, with [[H.sup.3]]-hypoxanthine incorporation by parasite culture incubated with drug for two times: 24 h and 72 h (Valentin et al., 1997). [IC.sub.50] were determined graphically in concentration versus percent inhibition curves.
Cytotoxicity and selectivity index
Toxicity of the different alkaloids was estimated on a human cell line, NIH 3T3 (fibroblasts) cultured under similar conditions to P. falciparum, except for 5% fetal calf serum instead of human serum. Subcultures were obtained by treatment with trypsine (0.125%) EDTA (0.02%) in PBS. For in vitro determination of the toxicity of the compounds, [[H.sup.3]]-hypoxanthine incorporation in the presence of drugs were compared with that of control cultures without chemical agents.
Selectivity index is represented by the ratio [IC.sub.50] cytotoxicity/ [IC.sub.50] activity (on resistant strains).
Combinatory antimalarial activity was determined as previously described by Martin et al., 1987. Briefly, growing subinhibitory dilutions of chioroquine were added with growing subinhibitory dilutions of alkaloid. After 24 h or 72 h of incubation parasite, growth inhibition was estimated by [[H.sup.3]]-hypoxanthine incorporation. Results of inhibition were analyzed by the construction of isobolograms (Gail Evans and Havkick, 1994).
Hemisynthesis of demethyl-aspidospermine
The demethylation of aspidospermine was performed according to a method described by Janot and Goutarel, 1957.
Results and Discussion
As shown in Table 1, most of the compounds showed a better antiplasmodial activity after incubation for 72 h. The [IC.sub.50] obtained suggested a ranking of the alkaloids into two different groups: The most active ones, 1-5, 7, 8, with [IC.sub.50] between 3.2 and 15.4 [micro]M and the less active ones, 9-12, with [IC.sub.50] between 22.6 and 52.6 [micro]M.
The values reported in Table 2 showed that the cytotoxicity is higher after 72 h of incubation. The selectivity index (SI) of the compounds 2, 3, 5 and 8 were more interesting, with values between 3.2 and 22.7, than compound 12 with a SI of 0.3.
For the evaluation of the chloroquine-potentiating activity of two alkaloids 3 and 5, isobolograms forming concave curves were observed (see Figures 1 and 2).
After examination of the results obtained for the antiplasmodial activity, two groups became apparent: the active alkaloids 1-5, 7, 8 and the less active ones, 9-12. From a structural point of view, 1-5, 7, 8 possessed a free ethyl side chain formed by C-i 8 and C- 19 while 9-12 exhibited a tetrahydrofuran ring including C-18 and C-19 and a phenol group at C-12. In the case of the first group, the substitution of the aromatic ring didn't seem to influence the activity. For the second group, it was interesting to know if the decrease of the activity was caused by the presence of the tetrahydrofuran ring or by the phenol group. Therefore, we have prepared by demethylation of aspidospermine 5 the compound 6 possessing a phenolic fonction and a free ethyl chain. The evaluation of its antiplasmodial activity (see Table 1) showed [IC.sub.50] close to the values obtained for the first group. This result suggested that the tetrahydrofuran ring may decrease the antiplasmodial effect.
In the case of the estimation of the selectivity index, compound 12 (SI = 0.3, 72 h) appeared to be the least interesting. This result confirmed us that the presence of the tetrahydrofuran ring decreased the antiplasmodial activity and increased the cytotoxicity.
Concerning the chloroquine-potentiating effect, Frappier et al., 1996, have demonstrated that the form of the isobologams indicate the modulation type: antagonism, agonism or additive effect of the compound tested on the inhibition produced by the chloroquine. In our present study, concave curves were obtained, which represent a potentiating effect.
These results suggested that the evaluation of the antiplasmodial activity should be extended to other aspidospermane alkaloids and derivatives, and to the vincadifformine-tabersonine skeleton. Furthermore, they may represent potential agents for the reversal of resistance to chloroquine.
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Table 1 In vitro antiplasmodial activity against P. falciparum of Aspidosperma indole Alkaloids. Alkaloids chloroquine-resistant (445 nM) [IC.sub.50] [micro]M [+ or -] sd 24 h 72 h 1 16.3 [+ or -] 2.9 3.8 [+ or -] 0.7 2 19.5 [+ or -] 7.2 3.2 [+ or -] 0.9 3 16.1 [+ or -] 3.0 5.6 [+ or -] 0.7 4 11.8 [+ or -] 0.9 4.1 [+ or -] 0.6 5 22.3 [+ or -] 11.6 5.6 [+ or -] 1.3 6 (c) 15.1 [+ or -] 1.9 12.2 [+ or -] 5.2 7 7.4 6.2 8 15.4 [+ or -] 4.2 12.7 [+ or -] 4.2 9 17.7 [+ or -] 4.9 28.5 [+ or -] 13.0 10 52.8 [+ or -] 7.1 25.6 [+ or -] 2.7 11 90.4 [+ or -] 43.7 59.2 [+ or -] 5.4 12 149.7 [+ or -] 27.6 49.5 [+ or -] 3.7 Alkaloids chloroquine-sensitive (79 nM) n (a) [IC.sub.50] [micro]M [+ or -] sd 24 h 72 h 1 4 11.0 [+ or -] 1.7 4.6 [+ or -] 0.5 2 3 13.1 5.1 3 3 22.0 [+ or -] 7.1 5.9 [+ or -] 1.5 4 2 9.3 [+ or -] 2.4 6.6 [+ or -] 1.4 5 4 nd (b) nd 6 (c) 4 21.5 [+ or -] 6.5 20.3 [+ or -] 6.2 7 1 34.0 15.4 8 3 27.2 8.7 9 3 40.8 [+ or -] 3.8 22.6 [+ or -] 2.5 10 2 113.1 55.3 11 3 44.4 28.0 12 2 169.3 57.3 Alkaloids n 1 3 2 1 3 3 4 3 5 6 (c) 3 7 1 8 1 9 2 10 1 11 1 12 1 (a)number of independent experiment, each done in triplicate (b)not determined (c)hemisynthetic alkaloid Table 2 Evaluation of the cytotoxicity and the selectivity index of some Aspidosperma indole alkaloids. Alkaloids cytotoxicity on NIH 3T3 [IC.sub.50][micro]M [+ or -] sd n (a) 24 h 72 h 2 129.8 [+ or -] 11.2 72.1 [+ or -] 2.2 2 3 127.4 [+ o r-] 6.8 87.1 [+ or -] 18.1 2 5 53.2 [+ or -] 0.8 46.2 [+ or -] 3.6 2 8 33.2 [+ or -] 7.6 40.8 [+ or -] 1.6 2 12 13.8 [+ or -] 4.4 13.0 [+ or -] 3.1 2 Alkaloids selectivity index (resistant strains) n 24 h 72 h 2 6.6 22.7 2 3 7.9 15.6 2 5 2.3 8.3 2 8 2.1 3.2 2 12 0.1 0.3 2 (a)number of independent experiment, each done in triplicate
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A.C. Mitaine-Offer (1), M. Sauvain (2), A. Valentin (3), J. Callapa (4), M. Mallie (3) and M. Zeches-Hanrot (1)
(1) Laboratoire de Pharmacognosie, Faculte de Pharmacie, Universite de Reims Champagne-Ardenne, Reims, France
(2) Institut de Recherche pour le Developpement, Paris, France
(3) Laboratoire d'Immunologie et Parasitologie, Faculte de Pharmacie, Universite de Montpellier I, Montpellier, France
(4) Instituto Boliviano de Biologia de Altura, Universidad Mayor de San Andres, La Paz, Bolivia
New address: A.-C. Mitaine-Offer, Laboratoire de Pharmacognosie, Unite MIB, J E 2244, Faculte de Pharmacie, Universite de Bourgogne, 7, Bd. Jeanne D'Arc, BP 87900, 21079 Dijon Cedex, France
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