Abstract
Context: With the emergence of strains multiresistant to antimalarial drugs, the search for new active molecules remains a priority. Ethnopharmacology appears to be a good method of selection in such investigations.
Objective: The aim of this research work is to select plants used in Melanesian traditional medicine, in New Caledonia and Vanuatu, which should be a promising source for the isolation of new antimalarial drugs.
Materials and methods: Forty-seven plant extracts belonging to 12 families, traditionally used by the Melanesian people or belonging to an antimalarial known genus, were screened in vitro for antimalarial activity on Plasmodium falciparum chloroquine (CQ)-resistant (FcB1) and CQ-sensitive (HB3) strains. They were also tested for their inhibitory effects on a protein kinase (Pfnek) and their cytotoxicity on human breast adenocarcinoma (MCF7) cells.
Results: Among all extracts, four displayed strong in vitro activities against P. falciparum: Gardenia urvillei Montrouzier, Scleria polycarpa Boeckeler, Terminalia catappa L. and Acronychia laevis J.R. & J.G. Forster, the latter being also toxic on MCF7 cells. Except for the extracts of S. polycarpa, all others that were active on P. falciparum, also possess an inhibitory effect on Pfnek.
Discussion and conclusion: These results confirm that ethnopharmacology is an excellent approach for such investigations. The two countries considered clearly present advantages in the field. Indeed, local populations keep their traditional knowledge alive, and their flora is exceptionally rich. In New Caledonia, the high endemicity rate (74%) ranks the island as one of the world’s biodiversity hotspots. As a consequence, chances to discover new active natural compounds are also high.
Introduction
New Caledonia is a French overseas territory in the South Pacific, about 2000 km east of Australia. The Republic of Vanuatu is an archipelago of 80 uninhabited islands, about 500 km NNE of New Caledonia. These two countries have a tropical climate characterized by a rainy season with an average temperature of 30°C from November to April and a dry season from May to October. According to CitationWHO (2005), malaria is present in 105 countries and territories, 10 of which are situated in the West Pacific, including Vanuatu. People living in countries with endemic malaria generally treat themselves with traditional remedies or use them as an alternative or complementary treatment (CitationWilcox & Bodeker, 2004). Since only a few plants from the South Pacific area have been investigated for antiplasmodial activity (CitationVerotta et al., 2001; CitationHay et al., 2004), we decided to search for novel natural bioactive antiplasmodial compounds within the flora of this region. New Caledonia and Vanuatu appear to be interesting lands for two reasons: on the one hand, local populations keep their traditional knowledge alive, on the other, the flora is exceptionally rich with a 74% endemicity rate in New Caledonia (CitationJaffre et al., 2001). Thus, the island is considered as one of the world’s hotspots of biodiversity. Consequently, chances to discover new active natural molecules are very likely.
Plant selection was based on ethnopharmacological data collected amongst local traditional healers. For biological evaluations we chose species which are traditionally used to treat fevers (the main symptom of malaria) and/or stomach troubles (a symptom frequently associated with malaria). Plant extracts were prepared and tested against Plasmodium falciparum in vitro culture. We also tested their capacity to inhibit Pfnek, a specific P. falciparum protein kinase involved in the completion of the parasite cycle (CitationReininger et al., 2005).
The pfnek-1 assay was chosen for two main reasons: firstly, there are no Plasmodium cultures in New Caledonia because of legal restrictions; secondly, because a direct link has been observed, in previous studies, between both antiplasmodial and Pfnek-1 activities (CitationLebouvier et al., 2009).
Materials and methods
Plant material
During a 15-year-long program on natural substances, carried out in Vanuatu and New-Caledonia, 17 native or endemic plants belonging to 12 families from New Caledonia and Vanuatu were selected on the basis of ethnopharmacological studies and bibliographical research. Species with strong medicinal indications generally used for fever treatment and gastro-intestinal disorders were selected (). Plants were collected in 2004 and herbarium voucher specimens were deposited at the Noumea IRD Herbarium (NOU). These latter were used by IRD botanists in order to identify species.
Table 1. Studied plants and their Melanesian traditional uses.
Selected plant parts () were sampled, oven-dried at 40°C for 48 h and ground before being immediately extracted by maceration. In the case of Terminalia catappa, leaves were also extracted fresh.
Table 2. Activity of plant extracts on HB3 and FCB1 Plasmodium falciparum strains, Pfnek and MC7 cells.
Preparation of plant extracts
All chemicals come from Sigma Aldrich (Saint-Quentin Fallavier, France). Dried powdered materials (60 g) were extracted by shaking maceration in 250 mL of ethanol (80%) or dichloromethane for 3 h at room temperature. The macerate was then dried and again extracted with methanol under the same conditions. The extracts were filtered and dried under reduced pressure.
Protein kinase inhibition test (Pfnek)
Antiplasmodial activity of extracts was first estimated with a protein kinase assay. Recombinant Pfnek-1 was purified from a transformed ampicillin-resistant bacteria Escherichia coli (BL21 strain) which produces a fusion protein carrying a glutathioneS-transferase fragment on one terminal end and the catalytic domain of Pfnek-1 on the other. The glutathioneS-transferase activity is used for enzyme purification by affinity chromatography on a glutathione resin (CitationDorin et al., 2001). The protein kinase activity was estimated by measuring 33P incorporation into β-casein, using γ-[33P]-ATP (Perkin Elmer, Courtaboeuf, France) as a phosphate donor. Test compounds were dissolved in DMSO and incubated in 20 mM Trizma pH 7.5, containing 20 mM MgCl2, 10 mM NaF and 10 µM ATP. β-Casein (3 mg/ mL) and γ-[33P]-ATP were introduced before the addition of 1 µg GST-Pfnek-1, which started the kinase reaction. 5 µCi of γ-[33P]-ATP were used per reaction. After incubation at 30°C for 30 min, each solution was transferred to a phosphocellulose filter paper (chromatography paper P81; Whatman-cation exchanger). After four successive washes with 1% H3PO4, the acid-precipitable radioactivity was measured using a liquid scintillation analyzer Packard 1600TR. The IC50, defined as the concentration of compounds inhibiting 50% of the enzyme activity, was estimated from the dose-response curves. Roscovitine (Sigma) was used as positive control.
In vitro assays on P. falciparum
Antiplasmodial activities were determined in vitro against the chloroquine-resistant (FCB1) and CQ-sensitive (HB3) strains of P. falciparum. P. falciparum was cultivated according to CitationTrager and Jensen (1976), on glucose-enriched RPMI 1640 medium supplemented with 10% human serum at 37°C. The original microdilution technique of CitationDesjardins et al. (1979) was then modified as described in CitationValentin et al. (1997). Plasmodium infected erythrocytes were plated at 3% parasitemia (5% hematocrit) with [3H]-hypoxanthine (Amersham, London) (0.8 µCi), in 96-well microtiter plates, and exposed to different concentrations of the crude extracts. Plates were incubated for 48 h, at 37°C in a 5% CO2 atmosphere. Microtiter plates were then frozen-defrosted and each well was harvested onto a glass fiber filter. At this point the incorporated [3H]-hypoxanthine was determined with a beta-counter (Perkin Elmer, Courtaboeuf, France). Controls were performed to assess the background (negative control) and the parasite growth (positive control). Growth curves (inhibition versus extract concentration) were plotted and the IC50 was determined graphically. Chloroquine was used as the inhibitor standard.
Results were classified according to CitationRasoanaivo et al. (2004): If the IC50 is less than 0.1 µg/mL, the activity was considered very good; from 0.1 to 5 µg/mL, good; from 5 to 10 µg/mL, moderate; over 11 µg/mL the extract was considered inactive.
Cytotoxicity assay
Human breast adenocarcinoma (MCF7) cells were grown in DMEM culture media containing 2 mM l-glutamine (Bio Wittaker) supplemented with 5% fetal calf serum (FCS) (Sigma Aldrich, Saint-Quentin Fallavier, France) and incubated under standard conditions (37°C, 5% CO2). All experiments were carried out using cells in the exponential growth phase. Cells were trypsinized, suspended in DMEM containing 5% FCS and seeded (40,000 cells/well) in 96-well plates. After 24 h, the medium was replaced by a fresh one containing various dilutions of the extract. Fresh complete medium without drugs was used as positive control. At the end of the treatment, cell viability was evaluated by measuring the mitochondrial enzyme succinate dehydrogenase activity. This test used sodium 3,3-1(1-phenyl-amino-carboxyl)-3-4-tetrazolium + bis-(4-methoxy-6-nitro)] benzene sulfonic acid hydrate] (XTT) as substrate which was converted to a formazan product detected spectrophotometrically at 450 nm (CitationJullian et al., 2005).
Doxorubicin was used as the control drug.
Results and discussion
Forty-seven extracts from 17 plants, belonging to 12 families were screened for antiplasmodial activity against Plasmodium falciparum infected erythrocytes and for Pfnek inhibition. Traditional uses are listed in and antiplasmodial activities are listed in . Only four extracts, Acronychia laevis J.R. & J.G. Forster (Rutaceae), Gardenia urvillei Montrouzier (Rubiaceae), Scleria polycarpa Boeckeler (Cyperaceae) and Terminalia catappa L. (Combretaceae), revealed to be active against all P. falciparum strains resistant or sensitive to chloroquine, with an IC50 < 10 µg/mL. Chloroquine used as control showed an IC50 of 120 nM against FCB1 and 36 nM against HB3 strains.
Nineteen extracts showed an inhibitory activity above 50% on Pfnek at a concentration of 25 µg/mL while roscovitine has an IC50 of 20 µM (7 µg/mL). Three extracts inhibited both P. falciparum in vitro growth and Pfnek, i.e., Gardenia urvillei, Terminalia catappa and Acronychia laevis. Only Acronychia laevis proved to be cytotoxic in the MCF7 model at 3 µg/mL while doxorubicin had an IC50 of 4 µM (2.3 µg/mL).
Acronychia laevis J.R. et J.G. Forster (Rutaceae)
Common in New Caledonia, Acronychia laevis is rarely used in traditional medicine as an antipyretic. Its vernacular name is “hmelexeci” in Drehu language (Lifou Island). According to CitationHorgen et al. (2001), species of Acronychia genus were considered as antiplasmodials. The extract of A. laevis stems inhibited Pfnek at 50.7% and had an IC50 of 5 µg/mL against FcB1 strain of P. falciparum, but the same extract was strongly cytotoxic against MCF7 cells at 3 µg/ml. The presence of quinoline and acridone alkaloids has been isolated from Acronychia species (CitationCui et al., 1999; CitationMichael, 2001). Such compounds could explain the observed antimalarial and cytotoxic activities of A. laevis.
Gardenia urvillei Montrouzier (Rubiaceae)
Gardenia urvillei is endemic to New Caledonia, commonly named “tiaré des forêts sèches” (dry forests “tiaré”). This plant is not considered as an antipyretic in traditional medicine in New Caledonia, but according to the literature the genus Gardenia contains some triterpenes with antiplasmodial activities, their IC50 ranging from 1.5 to 2.9 µg/mL (CitationEl-Tahir et al., 1999; CitationSuksamrarn et al., 2003). Ethanol extract of G. urvillei leaves inhibited the protein kinase (54.7%) and was active against P. falciparum (IC50 = 5 μg/ml) without toxicity on MCF7 cells at concentrations up to 100 μg/mL.
Scleria polycarpa Boeckeler (Cyperaceae)
Sometimes misnamed Scleria scrobiculata auct. plur., non Nees & Meyen in old publications, Scleria polycarpa can be found from Samoa and Tonga to Melanesia, northeast Australia, Marianas and Carolinas to West Malaysia (CitationSmith, 1979). Its vernacular name is “noyoteptep”in Motalava, Vanuatu, where the maceration of fresh flowers was used to treat hoarseness (CitationVienne, 1981–1982). Macerations of leaves were also recommended against fevers, diarrhea and gastro-intestinal problems in Indonesia (CitationGrosvenor et al., 1995). It also exists in Fiji where it is known by its local name, but Fijians do not use it in their traditional medicine (CitationSmith, 1979). The dichloromethane extract of dried leaves had no activity on the protein kinase (2.5% inhibition) but it showed an IC50 of 5 μg/mL against FcB1 strain of P. falciparum and was not cytotoxic against MCF7. A study on the medicinal plants used to treat malaria in Madagascar (CitationRasoanaivo et al., 1992) revealed that Scleria griegifolia Riedley leaves were used in decoctions in rural areas to treat fevers. This interesting activity observed with S. polycarpa may indicate the occurrence of potential antiplasmodial compounds in the genus Scleria. Moreover, the non-volatile compounds in the genus Scleria remain unknown. S. polycarpa is definitely a good candidate for further phytochemical investigations.
Terminalia catappa L. (Combretaceae)
T. catappa is an introduced species in New Caledonia and Vanuatu, commonly named “badamier”. People use this plant as an antipyretic, astringent, sudorific and antidiarrhetic (CitationRageau, 1973). According to CitationCambie and Ash (1994) a decoction of leaves or stems is also used against fevers and other symptoms in the South Pacific islands. Also, in Taiwan fallen leaves are used as an herb to treat liver diseases (CitationLin & Kan, 1990). In Suriname, a tea made from the leaves is prescribed against dysentery and diarrhea (CitationDeFilipps et al., 2004). It is also thought that the leaves contain antioxidant (CitationLin et al., 2001) and agents for prevention of cancers (although they have no demonstrated anticarcinogenic properties) (CitationMorioka et al., 2005) as well as chromosome breakage, and sickle cell anemia anticlastogenic characteristics (CitationMgbemene & Ohiri, 1999).
We showed that an ethanol extract of fresh leaves inhibited 60% of the protein kinase and 50% of P. falciparum growth at 7.7 μg/mL, without cytotoxicity against MCF7 cells at 100 μg/mL. T. catappa is known to contain high amounts of hydrolysable tannins (CitationLin et al., 2001). They could explain the antiplasmodial activity; indeed hydrolysable tannins isolated from Combretum molle (R. Br. Ex. G. Don.) Engl. & Diels showed a weak antiplasmodial activity (CitationAsres et al., 2001).
Conclusion
This study brings important insights to malaria research as the field is in constant need for alternative anti-malarial drugs. It appears to be the first attempt to screen antiplasmodial activities in plant extracts issued from New Caledonian flora. It concerned 17 medicinal species traditionally used for fever and gastro-intestinal disorder treatments, in New Caledonia or Vanuatu. Some of them showed both in vitro activities against Plasmodium falciparum and Pfnek and no cytotoxicity against MCF7 cells. Four were revealed to be very interesting, with important activities. Unfortunately, one of them, Acronychia laevis, was active but also cytotoxic. In contrast, three other plants of our sample, Gardenia urvillei, Scleria polycarpa and Terminalia catappa provide a foundation for further exploration of a new effective herbal drug and allow the isolation, and identification of antimalarial active compounds.
Acknowledgments
We thank the members of the Melanesian community (Vanuatu and the Thio council in NC) for sharing information about traditional practices and remedies. We are grateful to the members of Nouméa Herbarium for their advice. We also thank Elianne Pélissou [Unité mixte de recherche (mix unit of research) 152] and Alain Videault [Université de Nouvelle-Calédonie (University of New Caledonia)] for their technical assistance.
Declaration of interest
Edouard Hnawia, carried out several experiments at Unité mixte de recherche (mix unit of research), 152, thanks to a grant from Institut de Recherche pour le Développement (Research Institute for Development). The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
References
- Asres K, Bucar F, Knauder E, Yardley V, Kendrick H, Croft SL. (2001). In vitro antiprotozoal activity of extracts and compounds from the stem bark of Combretum molle. Phytother Res, 15, 613–617.
- Bril I. (2000). Dictionnaire nêlêmwa-nixumwak, français-anglais (Nouvelle-Calédonie). [nêlêmwa-nixumwak dictionnary, french-english (New Caledonia)] Collection Langues et Cultures du Pacifque [Pacific Languages and Cultures Collection], Selaf , 383, 523pp.
- Cabalion P, Waikedre J, Bontemps C, Degoy A, Fournet A. (2005). Substances naturelles de forêts sèches. Approche ethnobotanique des forêts de Baaba et Koumac. [ Natural substances of dry forest. Ethnobotanical approaches used for Baaba and Koumac forest] Programme Forêt Sèche de Nouvelle-Calédonie [Programm for New caledonian dry forest conservation] , 1, 117 pp.
- Cambie RC, Ash J. (1994). Fijian Medicinal Plants. CSIRO Australia.
- Cui B, Chai H, Dong Y, Horgen F, Hansen B, Maduli DA, Soejarto DD, Farnsworth NR, Cordell GA, Pezzuto JM, Kinghorn AD. (1999). Quinoline alkaloids from Acronychia laurifolia. Phytochemistry, 52, 95–98.
- DeFilipps RA, Maina SL, Crepin J. (2004). Medicinal Plants of the Guianas (Guyana; Surinam, French Guiana). Washington, DC: Department of Botany, National Museum of Natural History, Smithsonian Institution.
- Desjardins RE, Canfield CJ, Haynes JD, Chulay JD. (1979). Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique. Antimicrob Agents Ch, 16, 710–718.
- Dorin D, Le Roch K, Sallicandro P, Alano P, Parzy D, Poullet P, Meijer L, Doerig C. (2001). PfNek-1, a NIMA-related kinase from the human malaria parasite Plasmodium falciparum. Biochemical properties and possible involvement in MAPK regulation. Eur J Biochem, 268, 2600–2608.
- El-Tahir A, Satti GM, Khalid SA. (1999). Antiplasmodial activity of selected Sudanese medicinal plants with emphasis on Acacia nilotica. Phytother Res, 13, 474–478.
- Grosvenor PW, Gothard PK, McWilliam NC, Supriono A, Gray DO. (1995). Medicinal plants from Riau province, Sumatra, Indonesia. Part 1: Uses. J Ethnopharmacol, 45, 75–95.
- Hay AE, Helesbeux JJ, Duval O, Labaied M, Grellier P, Richomme P. (2004). Antimalarial xanthones from Calophyllum caledonicum and Garcinia vieillardii. Life Sci, 75, 3077–3085.
- Horgen FD, Edrada RA, de los Reyes G, Agcaoili F, Mudulid DA, Wongpanich V, Angerhofer CK, Pezzuto JM, Soejarto DD, Farnsworth NR. (2001). Biological screening of rain forest plot trees from Palawan Island (Philippines). Phytomedicine, 8, 71–81.
- Jaffre T, Morat P, Veillon JM, Rigault F, Dagostini G. (2001). Composition et caractérisation de la fore indigène de Nouvelle-Calédonie. [Composition and characterisation of the native flora of New Caledonia], Volume spécial II4, IRD, Noumea, New Caledonia.
- Jullian V, Bonduelle C, Valentin A, Acebey L, Duigou AG, Prevost MF, Sauvain M. (2005). New oxygenated clerodane diterpenoids from Laetia procera (Poepp.) Eichler (Flacourtiaceae), with antiplasmodial and antileishmanial activities. Bioorg Med Chem Lett, 15, 5065–5070.
- Lebouvier N, Jullian V, Desvignes I, Maurel S, Parenty A, Dorin-Semblat D, Doerig C, Sauvain M, Laurent D. (2009). Antiplasmodial activities of homogentisic acid derivative protein kinase inhibitors isolated from a Vanuatu marine sponge Pseudoceratina sp. Mar Drugs, 7, 640–653.
- Likhitwitayawuid K, Chanmahasathien W, Ruangrungsi N, Krungkrai J. (1998). Xanthones with antimalarial activity from Garcinia dulcis. Planta Med, 64, 281–282.
- Likhitwitayawuid K, Phadungcharoen T, Krungkrai J. (1998b). Antimalarial xanthones from Garcinia cowa. Planta Med, 64, 70–72.
- Likhitwitayawuid K, Dej-adisai S, Jongbunprasert V, Krungkrai J. (1999). Antimalarials from Stephania venosa, Prismatomeris sessiliflora, Diospyros montana and Murraya siamensis. Planta Med, 65, 754–756.
- Lin CC, Hsu YF, Lin TC. (2001). Antioxidant and free radical scavenging effects of the tannins of Terminalia catappa L. Anticancer Res, 21(1A), 237–243.
- Lin CC, Kan WS. (1990). Medical plants used for the treatment of hepatitis in Taiwan. Am J Chin Med, 18, 38–43.
- Mgbemene CN, Ohiri FC. (1999). Anti-sickling potential of Terminalia catappa leaf extract. Pharm Biol, 37, 152–154.
- Michael JP. (2001). Quinoline, quinazoline and acridone alkaloids. Nat Prod Rep, 18, 543–559.
- Morioka T, Suzui M, Nabandith V, Inamine M, Aniya Y, Nakayama T, Ichiba T, Yoshimi N. (2005). Modifying effects of Terminalia catappa on azoxiymethane-induced colon carcinogenesis in male F344 rats. Eur J Cancer Prev, 14, 101–105.
- Munguti K. (1994). Indigenous knowledge in the management of malaria and visceral leishmaniasis among the Tugen of Kenya. East Afr Med J, 75, 687–691.
- Ozanne-Riviere F. (1998). Le nyelâyu de Balade (Nouvelle-Calédonie). [ Nyelâyu from Balade], Edition Peters, Selaf, p. 112.
- Planchon L. (1894). Produits fournis à la matière médicale par la famille des Apocynaceae. [Products for medical practice coming from the Apocynaceae family]. Imprimerie centrale du Midi, Hamelin Frères, Montpellier, France.
- Rageau J. (1973). Plantes médicinales de la Nouvelle-Calédonie, Plantes fébrifuges. [Medicinal plants from New Caledonia. Antipyretic plants]. Travaux et documents de l’ORSTOM, Noumea, New Caledonia.
- Reininger L, Billker O, Tewari R, Mukhopadhyay A, Fennell C, Dorin-Semblat D, Doerig C, Goldring D, Harmse L, Ranford-Cartwright L, Packer J, Doerig C. (2005). A NIMA-related protein kinase is essential for completion of the sexual cycle of malaria parasites. J Biol Chem, 280, 31957–31964.
- Rasoanaivo P, Deharo E, Ratsimamanga-Urverg S, Frappier F. (2004). Guidelines for the evaluation of the non-clinical efficacy of traditional antimalarials. In: Wilcox M, Bodeker G, Rasoanaivo P, eds. Traditional Medicinal Plants and Malaria. CRC Press.
- Rasoanaivo P, Petitjean A, Ratsimamanga-Urverg S, Rakoto-Ratsimamanga A. (1992). Medicinal plants used to treat malaria in Madagascar. J Ethnopharmacol, 37, 117–127.
- Smith AC. (1979). Flora vitiensis nova. A new flora of Fiji (spermatophytes only). Volume 1. Lawai, Hawaii, Pacific Tropical Botanical Garden, 495 pp.
- Suksamrarn A, Tanachatchairatana T, Kanokmedhakul S. (2003). Antiplasmodial triterpenes from twigs of Gardenia saxatilis. J Ethnopharmacol, 88, 275–277.
- Tona L, Ngimbi NP, Tsakala M, Mesia K, Cimanga K, Apers S, De Bruyne T, Pieters L, Totte J, Vlietninck AJ. (1999). Antimalarial activity of 20 crude extracts from nine African medicinal plants used in Kinshasa, Congo. J Ethnopharmacol, 68, 193–203.
- Tona L, Cimanga RK, Mesia K, Musuamba CT, De Bruyne T, Apers S, Hernans N, Van Miert S, Pieters L, Totte J, Vlietninck AJ. (2004). In vitro antiplasmodial activity of extracts and fractions from seven medicinal plants used in the Democratic Republic of Congo. J Ethnopharmacol, 93, 27–32.
- Trager W, Jensen J. (1976). Human malaria parasites in continuous culture. Science, 193, 673–675.
- Valentin A, Benoit F, Moulis C, Stanislas E, Mallié M, Fourasté I, Bastide JM. (1997). In vitro antimalarial activity of pendulin, a bisbenzylisoquinoleine from Isopyrum thalictoides. Antimicrob Agents Ch, 41, 2305–2307.
- Verotta L, Dell’Agli M, Giolito A, Guerrini M, Cabalion P, Bosisio E. (2001). In vitro antiplasmodial activity of extracts of Tristaniopsis species and identification of the active constituents: Ellagic acid and 3,4,5-trimethoxyphenyl-(6′-O-galloyl)-O-beta-d-glucopyranoside. J Nat Prod, 64, 603–607.
- Vienne B. (1981-1982). Les usages médicinaux de quelques plantes AQ23 communes de la fore des Iles Banks (Vanuatu). [ Medicinal uses of common plants from Banks Island Flora (Vanuatu)]. Cahiers ORSTOM, 18, 569.
- WHO (2005). World malaria report. Geneva: World Health Organization. Available at: http://www.rbm.who.int/wmr2005/index.html (accessed October 2010)
- Wilcox ML, Bodeker G. (2004). Traditional herbal medicines for malaria. Br Med J, 329, 1156–1159.