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Des sources du savoir aux médicaments du futur

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Jacques Fleurentin
, 
Jean-Marie Pelt
, 
Guy Mazars

2. Études chimiques et pharmacologiques

Ethnopharmacology in the search for new leishmanicidal drugs

Bernard Weniger, A Estrada, R Aragón, E Deharo, G J Arango, A Lobstein et R Anton

Note de l’auteur

Note portant sur l’auteur1

Note portant sur l’auteur2

Note portant sur l’auteur3

Note portant sur l’auteur4

Note portant sur l’auteur5

Note portant sur l’auteur6

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Texte intégral

Introduction

1Cutaneous leishmaniasis is a zoonotic disease caused by species of the protozoal parasite, Leishmania. The disease causes deep characteristic tropical ulcers and/or nodules, which, upon healing, often result in disfiguring permanent scars. The different forms of leishmaniasis require expensive treatments, and the currently used medicines, pentavalent antimonials and/or pentamidine salts, show toxicity together with numerous side effects. Diverse cultural groups around the word have developed extensive inventories of ethnomedical therapies to treat parasitic infections. But, compared to malaria and other major tropical diseases, only a handful of authors have investigated the leishmaniasis-related ethnomedical knowledge, and practices or catalogued the medicinal plants used for treatment of the disease.

2The pacific coast of Colombia is part of the the biogeographical Chocó region which goes from Panama to the Ecuadorian coasts. The region is predominantly populated by black ethnic population and is an endemic area for malaria and cutaneous and mucocutaneous leishmaniasis. Traditional therapies against protozoal infections still play an important role among these communities.

Materials and methods

Ethnopharmacological and botanical studies

3Ethnopharmacological and botanical researches were carried out in the Department of Valle, in the Occidental part of Colombia, mainly on the Pacific coast near Buenaventura. The coastal area near Buenaventura, inhabited by Afro-Colombian communities and characterized by a very hot and humid climate, is occupied by primary and secondary forest and is a endemic region for cutaneous and mucocutaneous leishmaniasis. Besides selecting plants on the basis of ethnopharmacological criteria, we collected the other species on the basis of chemotaxonomic criteria. Herbarium samples were determined by Lic. R. Gonzalez, and voucher specimens were deposited at the Herbarium of the Universidad del Valle, Cali (CUVC).

Preparation of extracts

4For each part of plant, the methylene chloride extract was prepared by macerating 5 g of powdered dry plant material in stoppered flasks containing 50 ml of methylene chloride for 3 days. After extraction, the same plant material was dried and used again for the preparation of the methanolic extract, using 50 ml of methanol in a stoppered flask for 3 days. After filtration, the solvent was evaporated under reduced pressure.

Biological assays

5Leishmanicidal assays were performed in vitro on the promastigote forms of Leishmania. Three strains of Leishmania were used during these investigations: Leishmania mexicana amazonensis (IFLA/BR/67/PH8) responsible for the cutaneous form, L. braziliensis braziliensis (MHOM/BR/75/M 2903) responsible for the mucocutaneous form of the disease, and L. donovani infantum (MHOM/IN/PP75) responsible for the visceral form. All strains were obtained from IBBA (La Paz Bolivia).

6Leishmania promastigote were cultivated at 28°C. in Schneider-Drosofila medium (Sigma S9895) supplemented with heat inactivated (56°C. for 30 min) fetal calf serum (10%). Plant extracts passed through 0.22 µm Millipore filters, were previously dissolved in saline or DMSO (with a final concentration not exceeding 0.1 %) and then dissolved in the culture medium. Parasites in logarithm growth phase were dispatched in 96 flat bottom well plates at a concentration of 106/ml. Each well contained increasing concentration of the extract, from 10 µg/ml up to 100 µg/ml during 72 hours. The activity was determinated by evaluating the movements of the parasites with an inverted microscope and compared to control wells (without extract and with reference drugs). The movements were estimated as follow: 0 cross means that the parasite are in good conditions and the drug inactive; 1 cross, the drug is poorly active; crosses, the drug is active; 3 crosses, no movement is detected, the drug is very active. Pentamidine (Aldrich chemical) and ketoconazole (Janssen Pharmaceutical were used as reference drugs. All assays were carried out in triplicate (Moretti et al., 1998).

Results and discussion

7In table 1, we report the use of 5 plants used topically to treat cutaneous leishmaniasis on the Pacific coast of Colombia. Table 2 summarizes the results obtained with the extracts of the botanical species that showed toxicity against Leishmania spp.

84 out of the 5 species used traditionally against leishmaniasis (80 %) were active in vitro at 100 µmg/ml against Leishmania spp. promastigotes: Conobea scoparioides, Hygrophila guianensis, Otoba novogranatensis and Otoba parviflora. On the other hand, out of the 40 other species selected on the basis of bibliographic or chemotaxonomic criteria, 5 only (12 %) showed leishmanicidal activity in vitro: Tabernaemontana obliqua, Huberodendron patinoi, Protium amplium, Marila laxiflora and Guarea polymera.

9Hygrophila guianensis Nees (Acanthaceae), Chupador. The leaves of this herbaceous plant are used as a topical application against leishmaniasis by black and indigenous groups of Southwest Colombia (Caballero, 1995). Neither biological nor chemical data about this species could be found in the literature.

10Tabernaemontana obliqua (Miers) Leeuwenb. (Apocynaceae), syn. Bonafousia obliqua Miers, Mierda de guagua. Various species from this genus are used in Colombia and in all the Amazonian area as antirheumatic (García Barriga, 1992; Duke and Vasquez, 1994). The genus is well-known for the presence of indole alkaloids. Neither biological nor chemical data about this species could be found in the literature.

11Huberodendron patinoi Cuatrec. (Bombacaceae), Carrá. This large tree is used as a commercial source of timber on the Pacific coast of Colombia (Poyry, 1982). The species is endemic of the Chocó region. Neither biological nor chemical data about this species could be found in the literature.

12Protium amplium Cuatrec. (Burseraceae), Anime. Several species from this genus are sources of balsamic resinous latex used in Latin America against tumors and heavy colds (Pernet, 1972; Schultes and Raffauf, 1990). The resin essential oil of several Protium species, mainly constituted of monoterpenes and phenylpropanoids, show anti-inflammatory-related activity (Siani et al., 1999). Neither biological nor chemical data about P. amplum could be found in the literature.

13Marila laxiflora Rusby (Clusiaceae), Aceitillo. The genus Marila is distributed in the tropics of Central and South America and the West Indies. The roots of various species of this genus are used against dysentery by the Siona Indians of South Colombia (Schultes and Raffauf, 1990). Recently, antifungal xanthones were isolated from the roots of this species (loset et al., 1998).

14Guarea polymera Little (Meliaceae), syn. Guarea chalde Cuatrec., Chalde. This medium-large tree is used as a commercial source of timber on the Pacific coast of Colombia (Poyry, 1982). Neither biological nor chemical data about this species could be found in the literature.

15Otoba novogranatensis Moldenke (Myristicaceae), syn. Dialyanthera otoba (Humb. & Bonpl.) Warb., Otobo. The genus Otoba comprises about ten species of shrubs to tall trees native to upland areas from Costa Rica to the western Amazon and Venezuela (Schultes and Raffauf, 1990; Gentry, 1993). Neither biological nor chemical data about this species could be found in the literature.

16Otoba parviflora (Markgr.) A.H. Gentry (Myristicaceae), syn. Dialyanthera parvifolia Markgr., Otobo. The Waorani Indians from the Ecuadorian Amazon crush the bark and the red resin and rub it on the skin for treating infections caused by mites and fungi (Schultes and Raffauf, 1990). Farnesyl-homogentisic acid derivatives have been isolated from the seeds of the species (Ferreira et al., 1995).

17Conobea scoparioides (Cham. & Schltdl.) Benth. (Scrophulariaceae), Hierba de sapo. This aromatic herb or low shrub is also used in the Chocó region as anticonceptive (García Barriga, 1992). The aerial parts of the plant show cell adhesion inhibition in vitro, and contain cucurbitacin E and monoterpenes (Musza et al., 1994; Alpande de Morais et al., 1972).

Acknowledgment

18The authors wish to thank the informants from the Pacific area, R.T. Gonzalez for professional support, G. Ruiz and E. Balanza for technical assistance, COLCIENCIAS (project N°1115-05-353-96), the International Foundation for Sciences, the Ministère Français des Affaires Etrangères and ECOS-Nord (action n°CF99A01) for financial support.

Table I. Plant species used for leishmaniasis in Western Colombia

Table I. Plant species used for leishmaniasis in Western Colombia

AP : aerial part ; L : leaves ; R : root ; RE : resin-like bark exudate

Table II. In vitro leishmanicidal activity of plant extracts

(a) B: bark ; FR: fruits ; L: leaves; AP: aerial parts; S: seeds
(b) D: methylene chloride extract; M: methanol extract
(c) La: promastigotes of
Leishmania mexicana amazonensis (IFLA/BR/67/PH8);
Lb: promastigotes of
L. braziliensis braziliensis (MHOM/BR/75/M 2903);
Ld: promastigotes of
L. donovani infantum (MHOM/IN/PP75).
For La, Lb and Ld: 0 means that the drug is inactive, + that the drug is poorly active, ++ that the drug is active and +++ that the drug is very active at 100
μg/ml of extract
(d) V: voucher number.

Bibliographie

References

ALPANDE DE MORAIS A., MOURAO J.C., GOTTLIEB O.R., LEAO DA SILVA M., MARX M.C., MAIA J.G.S., MAGALHAES M.T (1972) Thymolcontaining amazonian essential oils, Anais da Academia Brasileira de Ciençias, 44: 315-318.

CABALLERO R. (1995) La etnobotánica en las comunidades negras e indígenas del delta del rio Patia, Quito, Ediciones Abya-Yala.

DUKE J.A., VASQUEZ R. (1994) Amazonian ethnobotanical dictionary, Boca Raton, CRC Press.

FERREIRA A.G, FERNANDES J.B., VIEIRA PC., GOTTLIEB O.R. (1995) Further farnesyl-homogentisic acid derivatives from Otoba parvifolia, Phytochemistry, 40: 1723-1728.

GARCIA BARRIGA H. (1992) Flora Medicinal de Colombia, Bogota, Tercer Mundo Editores.

GENTRY A.H. (1993) A field guide to the families and genera of woody plants of northwest South America, Washington DC, Conservation International.

IOSET J.-R., MARSTON A., GUPTA M.P., HOSTETTMANN K. (1998) Antifungal xanthones from the roots of Marila laxiflora, Pharmaceutical Biology, 36:103-106.

MORETTI C., SAUVAIN M., LAVAUD C., MASSIOT G., BRAVO J.A., MUÑOZ V. (1998) A novel antiprotozoal aminosteroid from Saracha punctata, Journal of Natural Products, 61:1390-1393.

MUSZA L.I., SPEIGHT P., MC ELHNEY S., BARROW C.J., GILLIUM A.M., COOPER R., KILLAR L.M. (1994) Cucurbitacins, cell adhesion inhibitors from Conobea scoparioides, Journal of Natural Products, 57: 1498-1502.

PERNET R. (1972) Phytochemistry of the Burseraceae, Lloydia, 35: 280-287.

POYRY J. (1982) Estudios generales del sector maderero en el Litoral Pacífico Colombiano, Cespedesia, 11: 7-67.

SCHULTES R.E., RAFFAUF R.F. (1990) The healing forest: medicinal and toxic plants of the Northwest Amazonia, Portland, Dioscorides Press.

SIANI A.C, RAMOS M.F.S., MENEZES-DE-LIMA JR. O., RIBEIRO-DOS-SANTOS R., FERNANDEZ-FERREIRA E., SOARES R.O.A., ROSAS E.C., SUSUNAGA G.S., GUIMARÃES A.C., ZOGHBI M.G.B., HENRIQUES M.G.M.O. (1999) Evaluation of anti-inflammatory-related activity of essential oils from the leaves and resin of species of Protium, Journal of Ethnopharmacology, 66: 57-69.

Notes de fin

1 Laboratoire de Pharmacognosie, Faculté de Pharmacie, Université Louis Pasteur de Strasbourg B.P. 24 67401 Illkirch Cedex (France) Email: weniger@pharma.u-strasbg.fr

2 Dep. de Química, Universidad del Valle A.A. 25360 Cali (Colombia) Email: raularan@quimica.univalle.edu.co

3 Dep. de Química, Universidad del Valle A.A. 25360 Cali (Colombia) Email: raularan@quimica.univalle.edu.co

4 Institut de Recherche pour le Développement (I.R.D.) CP 9214 La Paz (Bolivia) Email: Plantibba@megalink.com

5 Facultad de Química farmacéutica, Universidad de Antioquia Medellin (Colombia) Email: qjarango@quimbaya.udea.edu.co

6 Laboratoire de Pharmacognosie, Faculté de Pharmacie, Université Louis Pasteur de Strasbourg B.P. 24 67401 lllkirch Cedex (France) Email: weniger@pharma.u-strasbg.fr

7 Laboratoire de Pharmacognosie, Faculté de Pharmacie, Université Louis Pasteur de Strasbourg B.P. 24 67401 lllkirch Cedex (France) Email: weniger@pharma.u-strasbg.fr

Table des illustrations

Titre Table I. Plant species used for leishmaniasis in Western Colombia
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