In vitro antiplasmodial activity of 18 plants used in Congo Brazzaville traditional medicine

Journal of Ethnopharmacology 104 (2006) 168–174

In vitro antiplasmodial activity of 18 plants used in Congo Brazzaville traditional medicine S.F. Mbatchi a,b , B. Mbatchi a,d , J.T. Banzouzi a,c,∗ , T. Bansimba a , G.F. Nsonde Ntandou d , J.-M. Ouamba e , A. Berry b , F. Benoit-Vical b,f b

a Centre d’Etude et de Recherche M´ edecins d’Afrique (CERMA), B.P. 45, Brazzaville, Congo Service de Parasitologie et Mycologie, Hˆopital de Rangueil, CHU Toulouse, 1 Avenue Jean Poulhes, TSA 50032, 31059 Toulouse Cedex 9, France c Institut de Chimie des Substances Naturelles (CNRS), 1 Avenue de la Terrasse-Bat 27, 91198 Gif-sur-Yvette Cedex, France d Laboratoire de Biochimie et Pharmacologie, Facult´ e des Sciences de la Sant´e, Universit´e Marien NGOUABI, Brazzaville, B.P. 69, Congo e Unit´ e de Chimie du V´eg´etal et de la Vie, Facult´e des Sciences, Universit´e Marien NGOUABI, Brazzaville, B.P. 69, Congo f Laboratoire de Chimie de Coordination (CNRS), 205 Route de Narbonne, 31077 Toulouse Cedex 4, France

Received 1 February 2005; received in revised form 5 August 2005; accepted 29 August 2005 Available online 27 October 2005

Abstract Sixty-six extracts of 18 plants commonly used by traditional healers in Congo Brazzaville for the treatment of malaria have been investigated for in vitro antiplasmodial activity. Ethanolic and dichloromethane extracts of 7 among the 18 studied plants were moderately active (10 ␮g/ml < IC50 < 50 ␮g/ml). These extracts concerned Cassia siamea (bark), Cogniauxia podolaena (root), Landolphia lanceolata (root and leaves), Millettia versicolor (leaves), Pseudospondias microcarpa (leaves), Uapaca paludosa (leaves) and Vernonia brazzavillensis (leaves). These results support their traditional use as antimalarial plants. The bark extract of Uapaca paludosa showed a good activity (<10 ␮g/ml) and the extracts from Quassia africana (root and leaves) even exhibited IC50 values less than 1 ␮g/ml. Except for Quassia africana, for which the three solvents (water, ethanol and dichloromethane) present an effective extraction, no aqueous extract was highly active. The cytotoxicity of aqueous, DCM and ethanol extracts of Quassia africana was tested on KB cell lines. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Malaria; Medicinal plants; Pharmacological screening; Plasmodium falciparum; Traditional medicine

1. Introduction Despite all the efforts to eradicate malaria, this disease continues to be one of the greatest health problems facing the tropical and subtropical regions. WHO estimates that with 300–500 million clinical cases and more than 2 million deaths each year, malaria is one of the three most deadly communicable diseases in the world (WHO, 2003). The increasing prevalence of drug-resistant strains of Plasmodium falciparum, its most widespread etiological agent, to standard antimalarial drugs necessitates a continuous effort to search for new antimalarial drugs with new modes of action, used alone or in association. The search for new drugs can follow three directions: study of Plasmodium biochemical pathways, chemical

∗

Corresponding author. Tel.: +33 1 69 82 30 11. E-mail address: [email protected] (J.T. Banzouzi).

0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.08.068

synthesis and phytochemical investigation of medicinal plants. The third way has been chosen by the Centre d’Etudes et de Recherche de M´edecins d’Afrique (CERMA) and as a matter of fact, plants always have been a rich source of antiplasmodial compounds and many antimalarial drugs still in use today (quinoline and artemisinin-based antimalarials) were obtained from plant extracts or from structures designed by pharmacomodulation of the lead compounds. One of the most important missions of the CERMA is to promote the phytochemical exploration of African medicinal plants and of the traditional therapeutic know-how, in close cooperation with the traditional healers of our network. Indeed, Africa can boast of a consequent traditional pharmacopoeia (estimated at 30,000 medicinal species) and of a vast knowledge concerning the use of these plants, but the traditional preparations cannot be diffused without a preliminary validation in laboratory and an optimization of their formulation. Congo Brazzaville, located in one of the richest floristic zones of Africa (Guineo-Congolian

S.F. Mbatchi et al. / Journal of Ethnopharmacology 104 (2006) 168–174

zone) has an ancient cultural tradition of knowledge and use of medicinal plants. Among the plants used in this country for the treatment of malaria and its associated symptoms, we selected, in collaboration with the traditional healers, 18 plants entering the principal medications which they prescribe against this disease. Our aim was to evaluate in the first place their in vitro antiplasmodial activity before considering the study of the in vivo activity of the extracts, as well as the bio-guided isolation of the active compounds from the most active plants. The present study relates to the in vitro evaluation of the antiplasmodial activity of 66 extracts from 18 plants usually used in Congolese traditional medicine but little documented in the scientific literature. 2. Materials and methods

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50 ml of distilled water maintained at 100 ◦ C for 10 min. These preparations were left to cool for 4 h, until they were again at ambient temperature. They were first filtered on paper, then centrifuged for 15 min at 3000 rpm/min and finally freeze-dried, for a better conservation prior to their pharmacological study. The ethanolic and DCM extracts were prepared by maceration of the plant material in the organic solvent at room temperature (5 g of powder in 50 ml of solvent) for three successive periods of 2 days, with a change of solvent every 2 days. For each plant, the three extracts were collected, filtered and then concentrated under reduced pressure. Concerning the root of Quassia africana, the traditional preparation consisted of a simple maceration in cold water. We thus added this method to our tests, in order to allow a better comparison of the method used in the laboratory and that used by the traditional healers. The yields obtained for each extract are given in Table 1.

2.1. Selection and collection of plant material 2.3. Assay of in vitro antiplasmodial activity Ethnobotanical surveys carried out by M´edecins d’Afrique in Congo through the CERMA, in collaboration with our network of traditional healers, coupled to a systematic study of the literature and databases concerning the Congolese plants entering the preparations treating malaria and its associated symptoms gave us a preliminary list of 140 plants belonging to 55 families of angiosperms. In order to determine which plants we should study in priority, we ranked the plants following the application of five criteria: good knowledge of the plant by the traditional healers, few chemical studies of all kinds on the plant (<5), no studies concerning the antiplasmodial activity of the plant, no antiplasmodial compound identified, easy provisioning (known local name, presence located on the markets or known by traditional healers). This enabled us to select 18 plants. The 18 plants selected were collected in the South of Congo Brazzaville. The botanical names as well as the families to which the collected plants belong are listed in Table 1 . These plants were identified by the traditional healers who collected them, and the determination was validated by the botanists of the Centre d’Etude des Ressources V´eg´etales, Brazzaville, Congo (CERVE), by using the reference specimens of the Congo Brazzaville National Herbarium, their numbers being given in Table 1. A specimen of each collected plant was deposited at M´edecins d’Afrique. 2.2. Preparation of the extracts The collected plants were separated into one to four batches according to ethnobotanical indications: whole plant, leaves, root or bark (see Table 1). The various batches were dried separately at ambient temperature (25–30 ◦ C), protected from the light, on absorbing paper. The dry plants samples were ground to powder and stored at ambient temperature before extraction. For each plant, two types of extracts were prepared (water and ethanol) in accordance with the traditional conditions of use. The plants having shown the best activities were also extracted with dichloromethane (DCM), in order to extract the maximum active compounds for later studies. The aqueous extracts were obtained by decoction: 5 g of dried powder was treated with

A total of 66 extracts (28 aqueous, 27 ethanolic and 11 DCM extracts), representing 18 plants and 16 families, were screened for their antiplasmodial activity on human erythrocytes infected by FcM29-Cameroon, a chloroquine-resistant strain (IC50 of chloroquine = 175 nM) of Plasmodium falciparum. The parasites were maintained in vitro in human erythrocytes provided by the Bank of Blood (Toulouse, France), diluted to 1% haematocrit in RPMI 1640 medium (GIBCO BRL) supplemented with 5% of human serum. The samples to be tested were stored at +4 ◦ C before analysis. The ethanolic and DCM extracts were diluted beforehand in DMSO and then in RPMI (final 2% DMSO) while the aqueous extracts were directly diluted in RPMI. A series of 10-fold dilutions ranging from 0.01 to 100 ␮g/ml were prepared. The antiplasmodial activity of these extracts was evaluated by the radioactive microdilution method described by Desjardins et al. (1979) and modified as follows. Extract dilutions were tested three to five times in triplicate in 96-well plates with cultures at a parasitaemia of 1% and a haematocrit of 1%. For each test, the plates of parasite culture were incubated with plant extracts for 48 h and radioactive hypoxanthine was added to the medium 24 h after the beginning of incubation (Benoit-Vical et al., 1999). Parasite growth was estimated by [3 H]-hypoxanthine incorporation. Concentrations of extracts inhibiting 50% of the parasite growth (IC50 ) were determined graphically by plotting concentration versus percent inhibition. All tests were performed at least in duplicate. 2.4. Assay of cytotoxicity The cytotoxicity of Quassia africana root and leaf extracts was assayed against KB (human epidermoid carcinoma) cells grown in Dulbecco’s modified Eagle’s medium supplemented with 25 mM glucose, 10% (v/v) fetal calf serum, 100 UI penicillin, 100 ␮g/ml streptomycin and 1.5 ␮g/ml fungizone and kept under 5% CO2 at 37 ◦ C. Ninety-six-well plates were seeded with 500 KB cells per well in 200 ␮l medium. Twenty-four hours later, plant extracts dissolved in DMSO at a stock solution of 10 mg/ml were added for 72 h at a final concentration

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Table 1 Extracts obtained from the 18 selected plants Species

Families

Voucher number

Plant parts

Solvents

Yield (%)

Brillantaisia patula T. Anders.

Acanthaceae

Descoing, 9962

Whole plant

Water Ethanol

19 5

Buchholzia macrophylla Pax

Capparidaceae

Bouquet, 2456

Leaves

Water Ethanol

19 6

Cassia siamea Lam.

Caesalpiniaceae

Sita, 128

Bark

Water Ethanol Dichloromethane

7 3 2

Cogniauxia podolaena Baill.

Cucurbitaceae

Nere, 365

Whole plant

Water Ethanol Water Ethanol Dichloromethane

14 7 8 3 2

Root

Cyathula prostrata (L.) Blume

Amaranthaceae

Bouquet, 2002

Whole plant

Water Ethanol

14 5

Hua gabonii Pierre ex De Wild.

Celastraceae

Coquelin, 3899

Leaves

Water Ethanol Water

10 8 9

Water Ethanol Water Ethanol

11 14 14 11

Water Ethanol Dichloromethane Water Ethanol Dichloromethane

20 24 14 8 4 3

Water Ethanol Water Ethanol

11 6 13 11

Root Hymenocardia ulmoides Oliv.

Euphorbiaceae

Coquelin, 2371

Bark Leaves

Landolphia lanceolata (K. Schum.) Pichon

Apocynaceae

Nere, 1279

Leaves

Root

Markhamia sessilis Sprague

Bignoniaceae

Nere, 1370

Bark Leaves

Millettia versicolor Welw. ex Bak.

Fabaceae

Carlier, 199

Leaves

Water Ethanol Dichloromethane

11 6 4

Morinda morindoides (Bak.) Milne-Redh.

Rubiaceae

Bouquet, 108

Leaves

Water Ethanol Water Ethanol

12 5 8 5

Root Porterandia cladantha (K. Schum.) Keay

Rubiaceae

Nere, 2023

Leaves

Water Ethanol

22 23

Pseudospondias microcarpa (A. Rich.) Engl

Anacardiaceae

Bouquet, 216

Leaves

Water Ethanol Dichloromethane

19 15 11

Quassia africana Baill.

Simaroubaceae

Coquelin, 836

Leaves

Water Ethanol Dichloromethane Water Watera Ethanol Dichloromethane

13 22 7 33 40 14 9

Root

Scleria barteri Boeck.

Cyperaceae

Makany, 221

Whole plant

Water Ethanol

8 6

Tetracera alnifolia Willd.

Dilleniaceae

Sita, 1636

Leaves

Water Ethanol

18 21

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Table 1 (Continued ) Species

Families

Voucher number

Plant parts

Solvents

Yield (%)

Uapaca paludosa Aubr´ev. et L´eandri

Euphorbiaceae

Coquelin, 1517

Bark

Water Ethanol Dichloromethane Water Ethanol Dichloromethane

5 6 4 11 11 8

Water Ethanol Water Ethanol Dichloromethane Water Ethanol

6 8 12 11 7 10 11

Leaves

Vernonia brazzavillensis Aubr´ev. ex Comp`ere

Asteraceae

Descoing, 7051

Bark Leaves

Root a

Traditional preparation: maceration in cold water.

ranging from 0.01 to 50 ␮g/ml in a fixed volume of DMSO (1% final concentration). Controls received an equal volume of DMSO. The number of viable cells was measured with the MTS reagent (Promega, Madison, WI) and IC50 was calculated as the concentration of compound eliciting a 50% inhibition of cell proliferation. The tests were performed in duplicate. 3. Results and discussion The extraction yields for the various parts of the plants are presented in Table 1 and are classified by extraction solvent. It may be noted that, according to the plants, the strongest yields are obtained either with water, or with ethanol but never with DCM. The IC50 values obtained for the 66 extracts are presented in Table 2. For the purpose of this study, an IC50 value less than 10 ␮g/ml was classified as a high activity and an IC50 value between 10 and 50 ␮g/ml was considered as a moderate activity. Any extract having an IC50 > 50 ␮g/ml is regarded as having no activity. These results show that eight extracts presented high activities. These were the bark extracts of Uapaca paludosa and the leaves and root extracts of Quassia africana among which four extracts had very high IC50 values (<1 ␮g/ml). Sixteen extracts (nine ethanolic and seven DCM extracts) from the 18 studied plants exhibited moderate activities. In this case, the extracts concerned the bark of Cassia siamea, the roots of Cogniauxia podolaena, the root and leaves of Landolphia lanceolata, the leaves of Millettia versicolor, Pseudospondias microcarpa and Vernonia brazzavillensis and the bark and leaves of Uapaca paludosa. All this confirms their traditional use as antimalarial plants. The IC50 values of other extracts were more than 50 ␮g/ml and hence of no interest. Generally, the organic extracts (dichloromethane and ethanol) showed more interesting antiplasmodial activities than the aqueous extracts, with better results for dichloromethane than for ethanol. Ten plants (Brillantaisia patula, Buchholzia macrophylla, Cyathula prostrata, Hua gabonii, Hymenocardia ulmoides, Markhamia sessilis, Morinda morindoides, Porterandia cladantha, Scleria barteri and Tetracera alnifolia), though often used in traditional medicine, appeared to have little antiplasmodial activity. They are perhaps useful for treating associated symp-

toms, such as fever, or to enhance the immune system. This situation concerns plants such as Cinnamonum camphora, without activity against Plasmodium but with very high antipyretic properties (Benoit et al., 1996). We therefore have to find other tests to assay their activity. Another explanation for their lack of activity is perhaps the solvent used. We studied here only plants whose aqueous or ethanolic extracts exhibited an activity, because only these solvents are used in traditional medicine. However, we are well aware that plants showing no activity with these solvents can appear active against malaria when extracted with non-polar solvents, such as ether or chloroform. This seems to be the case for Morinda morindoides since the ether extract of this plant showed antiplasmodial activity in vitro as well as in vivo in preceding studies (Tona et al., 2001, 2004). Indeed, the best IC50 (1.8 ± 0.2 ␮g/ml) was obtained with an ether extract while the ethanolic extract had an IC50 superior to 50 ␮g/ml (94.2 ␮g/ml) (Tona et al., 2001). Morinda morindoides leaves have been evaluated for their in vivo antimalarial activity, in the 4-day-suppressive test against Plasmodium berghei ANKA in mice. The chemosuppression of parasitaemia, with oral administration (200 mg/kg), was 74% for the most active extract, obtained with DCM, and 30% for the ethanolic extract. Cassia siamea is administered per os in Congo, Benin, Togo and Sierra Leone to treat malaria, liver failure and fever (Guenoukpati, 1983; Kr¨uger, 1985; Adjanohoun, 1986, 1989). A similar study was carried out on the leaves of the same plant by a team working on the traditional medicine of Burkina-Faso (Sanon et al., 2003). This team used the strain W2, Plasmodium falciparum chloroquine-resistant and various types of extracts. They obtained IC50 = 23.15 ␮g/ml for the aqueous extracts, higher than 10 ␮g/ml for the extracts with chloroform, pure ethanol and 50% ethanol and between 4 and 10 ␮g/ml for alkaloids extracted from the leaves of the plant. These results confirm the preliminary study conducted by Gbeassor et al. (1989) which support our interest in this plant for the treatment of malaria and suggest a thorough study of the alkaloids present in the plant. Cogniauxia podolaena had never been studied for antiplasmodial activity. Congolese traditional medicine uses its roots in the treatment of malaria. It enters the medication of various par-

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Table 2 In vitro activity of the plant extracts on Plasmodium falciparum FcM29 (IC50 values in ␮g/ml) Species

Extracts

Water

Ethanol

Dichloromethane

Brillantaisia patula Buchholzia macrophylla Cassia siamea

Whole plant Leaves Bark

>100 >100 >100

>50 >50 31 ± 5

ND ND 21 ± 3

Cogniauxia podolaena

Whole plant Root

>100 86 ± 17

>50 26 ± 5

ND 21 ± 2

Cyathula prostrata

Whole plant

>100

>50

ND

Hua gabonii

Leaves Root

>100 >100

>50 ND

ND ND

Hymenocardia ulmoides

Bark Leaves

>100 >100

>50 >50

ND ND

Landolphia lanceolata

Leaves Root

>100 >100

28 ± 12 20 ± 7

17 ± 2 11 ± 4

Markhamia sessilis

Bark Leaves

>100 >100

>50 >50

ND ND

Millettia versicolor

Leaves

>100

33 ± 7

>50

Morinda morindoides

Leaves Root

>100 >100

>100 >100

ND ND

Porterandia cladantha Pseudospondias microcarpa

Leaves Leaves

60 ± 23 35 ± 10

>50 26 ± 10

ND 29 ± 6

Quassia africana

Leaves Root Roota

1.8 ± 0.9 2.2 ± 0.4 1.9 ± 0.1

0.8 ± 0.4 0.5 ± 0.1 –

0.9 ± 0.1 0.1 ± 0.07 –

Scleria barteri Tetracera alnifolia

Whole plant Leaves

>100 55 ± 9

>50 >50

ND ND

Uapaca paludosa

Bark Leaves

>100 >100

27 ± 6 36 ± 5

8±3 16 ± 4

Vernonia brazzavillensis

Bark Leaves Root Chloroquine (␮M)

>100 50 ± 17 >100 0.175

ND 22 ± 5 >50

ND 28 ± 10 ND

ND: not determined. a Traditional preparation: maceration in cold water.

asitic diseases, as potent antiseptic and febrifuge (information given by Congo Brazzaville traditional healers). Landolphia lanceolata is traditionally used in Congo for its antimalarial properties. Our study confirms the antiplasmodial activity of its leaves and roots. To our knowledge, this is the first study of this kind published both for this plant and for others of the same genus. Millettia versicolor had not yet been studied in the treatment of malaria, in contrast to Millettia usaramensis subspecies usaramensis (Yenesew et al., 2003a). This activity could be due to a rotenoid, usararotenoid C. Other rotenoids (deguelin, tephrosin) isolated from Millettia dura have a larvicide activity on the mosquito Aedes aegypti (Yenesew et al., 2003b). Other Millettia have chemoprotective (Shirwaikar et al., 2003), antipyretic (Srinivasan et al., 2003), anti-inflammatory (Yankep et al., 2003) and cytotoxic properties (Ito et al., 2000, 2004). Pseudospondias microcarpa has a moderate antiplasmodial activity. As yet, no representative of the genus had been studied

for the treatment of malaria, not even in the broader register of anti-infectious plants. Vernonia brazzavillensis, although not yet studied, belongs to a genus whose other representatives have been well studied, even for antimalarial properties. Most of them present sesquiterpenelactones (Lopes, 1991). Vernonia amygdalina exhibits antiplasmodial activity in vitro (Masaba, 2000; Alawa et al., 2003; Tona et al., 2004) as well as in vivo (Abosi and Raseroka, 2003) and contains many molecules: flavonoids, sesquiterpenoids, alkaloids, triterpenoids, glycosides and steroids. It is thought that triterpenes are responsible for the antiplasmodial activity of Vernonia brasiliana (Alves et al., 1997). Lastly, some Vernonia have antipyretic, analgesic, anti-inflammatory (Valverde et al., 2001; Iwalewa et al., 2003; Mazumder et al., 2003; Cioffi et al., 2004) and cytotoxic properties (Koul et al., 2003). Uapaca paludosa is the second particularly interesting plant in this study. Its activity was especially revealed by the DCM bark extract. The leaves also have an interesting antiplasmodial activity. To our knowledge, no study had yet been

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published on this plant. In traditional medicine, Uapaca paludosa enters antipyretic preparations (Bouquet, 1969). To this genus belongs Uapaca nitida, which has an antiplasmodial activity in vitro due to triterpenes and in particular to betulinic acid (Kirby et al., 1993; Steele et al., 1999). Finally, Uapaca togoensis shows an antibacterial activity in vitro (Kone et al., 2004). Quassia africana shows the highest antiplasmodial properties, whatever the part of plant or the solvent used for extraction. The DCM extract remains the most active, with IC50 value seldom met in the literature, ranging from 0.1 to 2.2 ␮g/ml, according to the part used and the mode of extraction. This plant therefore shows promise for malaria therapy. The Congolese traditional healers use per os an aqueous maceration of its roots and a decoction of its leaves. This plant contains simalikalactone D, a quassinoid known for its antiplasmodial activity (O’Neill et al., 1986), extracted in organic solvents. This compound was also identified in other species of the same genus, such as Quassia amara and Quassia undulata, which exhibit antiplasmodial activity in rats (Ajaiyeoba et al., 1999), as in Simaba guianensis whose antimalarial activity was shown in vitro (Cabral et al., 1993). It remains now to check by bio-assayed purification the chemical composition of the traditional preparations. Indeed, other quassinoids, such as samaderin, found in Quassia indica (Kitagawa et al., 1996) also have an antiplasmodial activity in vitro. It would therefore be advisable to seek them in Quassia africana. Quassinoids present many other additional properties particularly important during malaria crises: antiviral, as for simalikalactone D from the root of Quassia africana (Apers et al., 2002), anti-inflammatory and stimulative on the immune system, such as the samaderins (Kitagawa et al., 1996). Other Quassia species have been shown to have in vitro antibacterial and antifungal effects (Ajaiyeoba and Krebs, 2003). The cytotoxicity of Quassia africana extracts tested on KB cell lines are listed in Table 3. They ranged from 2.7 to 6.7 ␮g/ml (threeto nine-fold higher than IC50 against Plasmodium falciparum) for the leaves and the aqueous root extracts, and from 0.11 to 0.7 ␮g/ml for the other root extracts (near to IC50 against Plasmodium falciparum values). It seems to confirm our opinion that more than one antiplasmodial compound is present in Quassia africana extracts. Unfortunately, in the roots, the most effective active principle against Plasmodium does not show selective

Table 3 In vitro cytotoxicity of Quassia africana tested on KB cells Plant part

Solvent

Cytotoxicity (␮g/ml)

Cytotoxicity/ antiplasmodial activity ratio

Leaves

Water Ethanol Dichloromethane

6.7 5.4 6.1

3.7 6.8 6.8

Roots

Water Water (traditional preparation) Ethanol Dichloromethane

5.9 2.7

2.7 1.4

0.7 0.1

1.4 1.1

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activity against this parasite because it seems to have the same activity against KB cells. 4. Conclusion Among African traditional healers, plants have always been used for the treatment of malaria. However, without a scientific validation in laboratory, the traditional preparations cannot enter “modern” medicine. This study has highlighted eight promising plants for further antimalarial investigations, and the determination of their active constituents, with a view to rationalize and optimize their utilization. Moreover, since we studied only 18 plants of our preliminary list, from which many plants have not yet been investigated chemically or pharmacologically, this study supports, in our opinion, that plants remain a potential source of leads for drug development. Acknowledgements This work has been successfully carried out thanks to a multidisciplinary and multinational platform in which the teams of the CERMA and the Facult´e des Sciences of Brazzaville (Congo), the traditional healers of M´edecins d’Afrique network and a team of INSERM-CNRS (Toulouse, France) have taken part. We particularly wish to thank Drs. Christiane Poupat, Alain Ahond, Jean-Yves Lallemand of the Institut de Chimie des Substances naturelles (CNRS, Gif-sur-Yvette, France), Adrien Cav´e of the Centre de Biochimie Structurale (CNRS, Montpellier, France), Jean-Franc¸ois Magnaval (Rangueil Hospital University, Toulouse) and Bernard Meunier (LCC-CNRS, Toulouse) who have supported our project. We also thank Mrs. Genvi`eve Aubert of Institut de Chimie des Substances naturelles (CNRS, Gif-sur-Yvette, France) for the cytotoxicity tests, all the members of M´edecins d’Afrique who participated in this work (particularly Miss Aline Prost), the traditional healers who collected the plants and supported us daily in our work, and the botanists of the CERVE for the validation of the botanical determinations. References Abosi, A.O., Raseroka, B.H., 2003. In vivo antimalarial activity of Vernonia amygdalina. British Journal of Biomedical Sciences 60, 89–91. Adjanohoun, E.J. (dir.), 1986. Contribution aux e´ tudes ethnobotaniques et floristiques au Togo. Agence de coop´eration culturelle et technique, A.C.C.T., Paris, 671 pp. Adjanohoun, E.J. (dir.), 1989. Contribution aux e´ tudes ethnobotaniques et floristiques au B´enin. Agence de coop´eration culturelle et technique, A.C.C.T., Paris, 895 pp. Ajaiyeoba, E.O., Abalogu, U.I., Krebs, H.C., Oduola, A.M., 1999. In vivo antimalarial activities of Quassia amara and Quassia undulata plant extracts in mice. Journal of Ethnopharmacology 67, 321–325. Ajaiyeoba, E.O., Krebs, H.C., 2003. Antibacterial and antifungal activities of Quassia undulata and Quassia amara extracts in vitro. African Journal of Medical Sciences 32, 353–356. Alawa, C.B., Adamu, A.M., Gefu, J.O., Ajanusi, O.J., Abdu, P.A., Chiezey, N.P., Alawa, J.N., Bowman, D.D., 2003. In vitro screening of two Nigerian medicinal plants (Vernonia amygdalina and Annona senegalensis) for anthelmintic activity. Veterinary Parasitology 113, 73–81.

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