Printer Friendly

Antiplasmodial activity of Aspidosperma indole alkaloids.

Summary

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

Introduction

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

Culture conditions

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).

Antiplasmodial activity

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).

Chloroquine-potentiating activity

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.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]
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


References

Desjardins, R.E., Canfield, C.J., Haynes, J.D., Chulay, J.D.: Quantitative assessment of antimalarial activity in vitro by a semi-automated microdilution technique. Antimicrob. Agents Chemother. 16: 710-718, 1979.

Frappier, F., Jossang, A., Soudon, J., Calvo, F, Rasoanaivo, P., Ratsimamanga-Urverg, S., Saez, J., Schrevel, J., Grellier, P.: Bisbenzylisoquinolines as Modulators of Chloroquine Resistance in Plasmodium falciparurn and Multidrug Resistance in Tumor Cells. Antimicrob. Agents Chemother. 40: 1476, 1996.

Frederich, M., Hayette, M.-P., Tits, M., De Mol, P., Angenot, L.: In Vitro Activities of Strychnos Alkaloids and Extracts against Plasmodium falciparum. Antimicrob. Agents Chemother. 43: 2328-2331, 1999.

Gail Evans, S., Havkick, I.: In vitro drug interaction between amantadine and classical antimalarial drugs in Plasmodiumfalciparum infections. Trans. R. Soc. Trop. Med. Hyg. 88: 683-686, 1994.

Iwu, M.M., Klayman, D.L., Jackson, J.E., Tally, J.D., Andersen, S.L.: Alkaloids of Picralima nitida used for treatment of protozoal diseases. The Secretary of the Army USA Patent, 1994, N[degrees] 5,290, 553, 21 p.

Janot, M.M., Goutarel, R.: Derivatives of the ibogaine alkaloids. Les Laboratoires Gobet, USA patent, 1957, 2,813,873, 3 p.

Martin, S.K., Oduola, A.M.J., Milhou, W.K.: Reversal of chloroquine resistance in Plasmodium falciparum by verapamil. Science 235: 899-901, 1987.

Mitaine, A.-C., Mesbah, K., Petermann, C., Arrazola, S., Moretti, C., Zeches-Hanrot, M., Le Men-Olivier, L.: Alkaloids from Aspidosperma Species from Bolivia. Planta Med. 62: 458-461, 1996.

Mitaine, A.-C., Weniger, B., Sauvain, M., Lucumi, E., Aragon, R., Zeches-Hanrot, M.: Indole Alkaloids from the Trunk Bark of Aspidosperma megalocarpon. Planta Med. 64: 487, 1998.

Mustofa, Valentin, A., Benoit-Vical, F., Pelissier, Y, KoneBamba, D., Mallie, M.: Antiplasmodial activity of plant extracts used in west African traditional medicine. J. Ethnopharmacol. 73: 145-151, 2000.

Trager, W., Jensen, J.: Human malaria parasite en continuous culture. Science 193: 673-675, 1976.

Valentin, A., Benoit-Vical, F., Moulis, C., Stanislas, E., Mallie, M., Fouraste, I., Bastide, J.-M.: In vitro antimalarial activity of penduline, a bisbenzylisoquinoline from Isopyrum thalictroides. Antimicrob. Agents Chemother. 41: 2305-2307, 1997.

Wright, C.W., Allen, D., Cai, Y, Phillipson, J.D., Said, I.M., Kirby, G.C., Warhurst, D.C.: In vitro Antiamoebic and Antiplasmodial Activities of Alkaloids Isolated from Alstonia angustifolia Roots. Phytother. Res. 6:121-124, 1992.

Wright, C.W., Allen, D., Cai, Y, Chen, Z., Phillipson, J.D., Kirby, G.C., Warhurst, D.C., Tits, M., Angenot, L. : Selective Antiprotozoal Activity of Some Strychnos Alkaloids. Phytother. Res. 8: 149-152, 1994.

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

Address

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

Tel.: ++33-3-80 39 32 36; Fax: ++33-3-80 39 33 00; e-mail: acoffer@u-bourgogne.fr
COPYRIGHT 2002 Urban & Fischer Verlag
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2002 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Author:Mitaine-Offer, A.C.; Sauvain, M.; Valentin, A.; Callapa, J.; Mallie, M.; Zeches-Hanrot, M.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Date:Mar 1, 2002
Words:2085
Previous Article:Antinociceptive, anti-inflammatory and acute toxicity effects of Zhumeria majdae extracts in mice and rats.
Next Article:Structure-antiemetic-activity of some diarylheptanoids and their analogues.


Related Articles
Studies on medicinal plants of Ivory Coast: investigation of Sida acuta for in vitro antiplasmodial activities and identification of an active...
Comparative antiplasmodial, leishmanicidal and antitrypanosomal activities of several biflavonoids.
Acetylcholinesterase inhibitors from plants.
Antileishmanial activity of indole alkaloids from Aspidosperma ramiflorum.
In vitro antiplasmodial activity of extract and constituents from Esenbeckia febrifuga, a plant traditionally used to treat malaria in the Brazilian...
Utilitarian to upscale: Peroba rosa is on a straight and narrow path to greater popularity.

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters