Annals of Tropical Medicine & Parasitology, Vol. 104, No. 5, 391–398 (2010) Antiprotozoal activities of some constituents of Markhamia tomentosa (Bignoniaceae) F. TANTANGMO*, B. N. LENTA{, F. F. BOYOM{, S. NGOUELA*, M. KAISER1, E. TSAMO*, B. WENIGER", P. J. ROSENTHAL** and C. VONTHRON-SÉNÉCHEAU",{{ * Department of Organic Chemistry, Faculty of Science, TWAS Research Unit of the University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon { Department of Chemistry, Higher Teachers’ Training College, University of Yaoundé I, P.O. Box 47, Yaoundé, Cameroon { Department of Biochemistry, Faculty of Science, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon 1 Swiss Tropical and Public Health Institution, Socinstrasse 57, CH-4002 Basel, Switzerland " Laboratoire de Pharmacognosie et Molécules Naturelles Bioactives, UMR 7200, Faculté de Pharmacie, Université de Strasbourg, B.P. 60024, 67401, Illkirch Cedex, France ** Division of Infectious Diseases, Department of Medicine, University of California, San Francisco, 1001 Portero Avenue, San Francisco, CA 94943, U.S.A {{ Laboratoire de Biologie et de Biotechnologies Marines, UMR M IFREMER 100, Université de Caen Basse-Normandie, Esplanade de la Paix, 14032 Caen Cedex, France Received 10 May 2010, Accepted 17 May 2010 Phytochemical investigation of an ethyl-acetate extract of the stem bark of Markhamia tomentosa (Bignoniaceae), which had good antimalarial activity in vitro, resulted in the isolation of eight known compounds: 2acetylnaphtho[2,3-b]furan-4,9-dione (1), 2-acetyl-6-methoxynaphtho[2,3-b]furan-4,9-dione (2), oleanolic acid (3), pomolic acid (4), 3-acetylpomolic acid (5), tormentic acid (6), b-sitosterol (7) and b-sitosterol-3-O-b-Dglucopyranoside (8). The structures of these compounds were established by spectroscopic methods. Each of compounds 1, 2, 4 and 5 was evaluated in vitro for its antiprotozoal activities against the ring stages of two chloroquine-resistant strains of Plasmodium falciparum (K1 and W2), the amastigotes of Leishmania donovani, and the bloodstream trypomastigotes of Trypanosoma brucei rhodesiense (the species responsible for human malaria, visceral leishmaniasis and African trypanosomiasis, respectively). Although compounds 1 and 2 exhibited potent antiprotozoal activities, they also showed high toxicity against a mammalian (L-6) cell line. Plants of the genus Markhamia are widely distributed in Africa, where they are used for the treatment of several human diseases (Kerharo, 1978; Letouzey, 1982; Adjanouhoun et al., 1996). In Tanzania, for example, aqueous extracts of the root bark of M. lutea are used to treat anaemia and diarrhoea (Kerharo, 1978; Adjanouhoun et al., 1996). Reprint requests to: C. Vonthron-Sénécheau. E-mail: [email protected]. # W. S. Maney & Son Ltd 2010 DOI: 10.1179/136485910X12743554760180 In Cameroon, M. lutea and M. tomentosa are both used to cure various microbial and parasitic diseases (Adjanouhoun et al., 1996). In previous phytochemical studies of Markhamia spp, bio-active lignans, phenyl glycosides, anthraquinones, alkaloids, phenyl propanoids and terpenoids have been isolated (Adesanya and Nia, 1997; Kernan et al., 1998; Khan and Mlungwana, 1999; Kanchanapoom et al., 2002; Lacroix et al., 2009). As part of an ongoing investigation of antiprotozoal agents in Cameroonian medicinal plants, an 392 TANTANGMO ET AL. ethyl-acetate extract of M. tomentosa was prepared and found to have good antimalarial activity in vitro against two strains (K1 and W2) of Plasmodium falciparum (see below). Chromatographic fractionation of this extract led to the isolation of eight known compounds: 2-acetylnaphtho[2,3-b] furan-4,9-dione (1), 2-acetyl-6-methoxynaphtho[2,3-b]furan-4,9-dione(2), oleanolic acid (3), pomolic acid (4), 3-acetylpomolic acid (5), tormentic acid (6), b-sitosterol (7) and b-sitosterol-3-O-b-D-glucopyranoside (8). The preparation of three extracts of M. tomentosa stem bark, the isolation of compounds 1–8 from the ethyl-acetate extract, and evaluation of the in-vitro activities of the extracts and some of the isolated compounds, against strains of Plasmodium, Leishmania and Trypanosoma and a mammalian cell line, are described below. MATERIALS AND METHODS General Melting points were determined on an M-540 melting-point unit (Büchi, Flawil, Switzerland). Optical rotations were measured, in chloroform solution, on a DIP-3600 digital polarimeter (JASCO, Tokyo). Infra-red (IR) spectra were determined on a Fourier transform IR spectrometer (Jasco) while ultra-violet (UV) spectra were determined on a Unicam spectrophotometer (Spectronic Analytical Instruments, Leeds, U.K.). 1H and 13C nuclear-magnetic-resonance (NMR) spectra were investigated on a Bruker spectrometer (Bruker Optik, Ettlingen, Germany) equipped with 5-mm 1H and 13C probes operating at 500 and 125 MHz, respectively, with tetramethylsilane as the internal standard. Silica gels of 230- to 400-mesh and 70- to 230-mesh (Merck, Darmstadt, Germany) were used for flash and column chromatography, respectively, while aluminium sheets precoated with silica gel 60 F254 (Merck) were used for thin-layer chromatography (TLC), with various mixtures of petroleum ether, nhexane, ethyl acetate, and acetone as eluents. Spots were visualized with UV light (at 254 and 365 nm) or using methanol–H2SO4 reagent. Plant Material The stem bark of M. tomentosa was collected in June 2007 at Mont Eloundem (Yaoundé) in the Central province of Cameroon. The plants were identified by N. Victor, a botanist at the National Herbarium of Cameroon, where a voucher specimen (6475/SRF/Cam) was deposited. Extraction and Isolation The dried stem bark (4 kg) of M. tomentosa was extracted successively with hexane, ethyl acetate and methanol, by maceration. In each extraction, two 10-litre volumes of solvent were used over a period of 48 h. After concentration under vacuum at room temperature, a green hexane (9 g), a brown ethyl-acetate (50 g) and a brown methanolic extract (100 g) were produced. When each of these extracts was screened in vitro for its activities against the W2 and K1 strains of Plasmodium falciparum, the ethyl-acetate extract was found to have the highest antimalarial activity against both strains (see below). This extract was therefore fractionated by column chromatography on silica gel (230- to 400-mesh), with nhexane–ethyl-acetate mixtures of increasing polarity being used as the eluents. Ninety fractions, each of 400 ml, were collected and combined, on the basis of the results of TLC, to yield four main fractions that were labelled F1 (4.0 g), F2 (7.1 g), F3 (9.3 g) and F4 (11.7 g). Fraction F1 (4.0 g) was essentially an oil that was not further investigated. Fraction F2 (7.1 g) was subjected to column chromatography over silica gel (70- to 230mesh), eluting with n-hexane–ethyl-acetate gradient mixtures. This resulted in the collection of 76 sub-fractions, each of 150 ml, which were combined on the basis of the results of TLC analysis. Further purification of sub-fractions 40–45 afforded ANTIPROTOZOAL ACTIVITIES OF Markhamia b-sitosterol 7 (300 mg) and 2-acetylnaphtho[2,3-b]furan-4,9-dione 1 (30 mg). Sub-fractions 53–55 yielded 2-acetyl-6methoxynaphtho[2,3-b] furan-4,9-dione 2 (35 mg). Successive chromatography of fractions 68–70 afforded oleanolic acid 3 (625 mg). Fraction F3 (9.3 g) was also subjected to column chromatography over silica gel (70- to 230-mesh), eluting with n-hexane– ethyl-acetate mixtures (80 : 20–75 : 25) to yield pomolic acid 4 (200 mg) and a powder of a mixture of compounds (400 mg). The powder was rechromatographed, using silica gel (70- to 230-mesh) and eluting with dichloromethane–methanol mixtures (99 : 1–98.5 : 1.5), to yield 3acetylpomolic acid 5 (300 mg), tormentic acid 6 (30 mg) and b-sitosterol-3-O-b-Dglucopyranoside 8 (600 mg). Fraction F4 (11.7 g) was another complex mixture that was not studied further. Assays of Biological Activity ANTIMALARIAL ACTIVITY (W2 STRAIN) Antimalarial activity was first determined, in vitro, using the W2 strain of P. falciparum, which is resistant to chloroquine and some other antimalarial drugs (Singh and Rosenthal, 2001). The parasites were cultured in sealed flasks at 37uC, in an atmosphere containing 3% (v/v) O2, 5% (v/v) CO2 and 91% (v/v) N2, in RPMI 1640 medium with 25 mM HEPES (pH 7.4), 10% (v/v) heat-inactivated human serum, and sufficient human erythrocytes to achieve a 2% haematocrit. Parasites were synchronized at the ring stage, by serial treatment with 5% (v/v) sorbitol (Sigma; Lambros and Vanderberg, 1979) and studied at 1% parasitaemia. Each of compounds 1, 2, 4 and 5 was prepared as a 10-mM stock solution in dimethyl sulphoxide (DMSO) and diluted as needed for the individual experiments, with each test dilution tested in triplicate. The stock solutions were diluted with HEPES- and serum-supplemented RPMI 1640 medium to give (0.2% (v/v) DMSO. Each test 393 dilution was gently mixed with an equal volume of parasite culture (showing 1% parasitaemia and at a 4% haematocrit). Negative controls contained the same concentrations of DMSO but no test compound whereas the cultures used as positive controls contained 1 mM chloroquine phosphate (Sigma) and no test compound. Once set up, the test cultures were incubated at 37uC for 48 h (representing one cycle of erythrocytic invasion and intra-erythrocytic multiplication) before being fixed by replacing the medium with an equal volume of 1% (w/v) formaldehyde in 0.1 M phosphatebuffered saline at pH 7.2 (PBS). Aliquots (50 ml) of each fixed culture were then added to 5-ml round-bottomed polystyrene tubes, each of which contained 0.5 ml PBS holding 0.1% (v/v) Triton X-100 and 1.0 nM YOYO nuclear dye (Molecular Probes, Eugene, OR). Parasitaemias in the test and control cultures were then compared using a flow cytometer (FACSortTM; BD, Franklin Lakes, NJ) to count the nucleated (i.e. parasitised) erythrocytes in each sample. The counts were recorded using the CellQuestTM software package (BD), with the test-culture counts normalized to percentages of the corresponding counts for the positive-control cultures. (K1 STRAIN) The antimalarial activity of each crude extract and each of compounds 1, 2, 4 and 5 was also assessed quantitatively, in vitro, using the microculture radio-isotope technique described by Desjardins et al. (1979), as modified by Ridley et al. (1996). The assay uses the uptake of [3H]hypoxanthine by parasites as an indicator of viability. Continuous in-vitro cultures of the asexual erythrocytic stages of the pyrimethamineand chloroquine-resistant K1 strain of P. falciparum (Thaithong and Beale, 1981) were maintained following the methods of Trager and Jensen (1976). Each extract or compound was tested after two-fold serial dilution, at seven concentrations between 20 and 0.31 mg/ml. After incubation of the ANTIMALARIAL ACTIVITY 394 TANTANGMO ET AL. parasites with the extract/compound for 48 h at 37uC, [3H]hypoxanthine (Amersham International, Little Chalfont, U.K.) was added to each well and the incubation was continued for another 24 h at the same temperature. Chloroquine (Sigma) was again used as a positive reference. LEISHMANICIDAL ACTIVITY For the in-vitro assays of leishmanicidal activity, 50 ml of culture medium — a 1 : 1 mixture of SM medium (Cunningham, 1977) and SDM-79 medium (Brun and Schönenberger, 1979) at pH 5.4, supplemented with 10% (v/v) heat-inactivated foetal calf serum (FCS) — was added to each well of a 96-well microtitre plate (Costar, Cambridge, MA). Serial dilutions of a crude extract, compounds 1, 2, 4 or 5 or the reference drug (miltefosine; Zentaris, Frankfurt, Germany) were prepared in duplicate, in the wells, to give 50 ml/well and concentrations between 30 and 0.041 mg/ml. Then 105 axenicallygrown amastigotes (Bates, 1993) of Leishmania donovani (MHOM/ET/67/L82) in 50 ml medium were added to each well, before the plate was incubated at 37uC, under a 5%-CO2 atmosphere, for 72 h. A 12.5% (w/v) aqueous solution of resazurin was then added, at 10 ml/well, and incubation continued for a further 2–4 h. The plate was then read in a microplate fluorometer (Spectramax Gemini XS; Molecular Devices, Sunnyvale, CA), using an excitation wavelength of 536 nm and an emission wavelength of 588 nm (Raz et al., 1997). Fluorescence development was measured and expressed as a percentage of the corresponding positivecontrol (miltefosine) value. ANTITRYPANOSOMAL ACTIVITY The procedures described by Freiburghaus et al. (1996) were used to test the in-vitro activity of each crude extract and isolated compounds 1, 2, 4 and 5. Working stock solutions (of 180 mg/ml) were prepared in the rabbit-serum-containing culture medium described by Baltz et al. (1985) and dispensed, at 100-ml/well, into the first row of wells of a 96-well microtitre plate (Costar). Complete culture medium was then added to the other wells, so that threefold serial dilutions of each extract/compound could be prepared. After the addition to each well of 26103 of the bloodstream forms of Trypanosoma brucei rhodesiense, from axenic culture (Baltz et al., 1985), the concentration of the extract/compound in each 100-ml culture ranged from 90 to 0.13 mg/ml. After incubation for 72 h in a humidified atmosphere at 37uC, with 5% (v/v) CO2, parasite development was assessed with resazurin — as for L. donovani but incubating with the resazurin for 24 h and using melarsoprol (ArsobalH; Rhône Poulenc Rorer, Paris), not miltefosine, as the reference drug. CYTOTOXICITY The cytotoxicity of each crude extract and compounds 1, 2, 4 and 5 was assessed using the L-6 cell line (of rat skeletal-muscle myoblasts) and the method of Pagé et al. (1993) as modified by Ahmed et al. (1994). The rat cells were seeded in 96-well microtitre plates (Costar) to give 103 cells in 50 ml complete medium [MEM supplemented with 10% (v/v) heat-inactivated FCS] in each well. A three-fold serial dilution of an extract or isolated compound, prepared in the complete culture medium, was then added, at 50 ml/well, to give final concentrations of 90 to 0.13 mg extract or compound/ml. Plates were then incubated at 37uC for 72 h in a humidified incubator, with 5% (v/v) CO2. Resazurin was again added as a viability indicator (Ahmed et al., 1994), incubating with the resazurin for 2 h before each plate was ‘read’ on the fluorescence scanner. Podophyllotoxin (Polysciences Inc., Warrington, PA) was used as the reference drug. DATA ANALYSIS Median inhibitory concentrations (IC50) were calculated, for each extract and iso- ANTIPROTOZOAL ACTIVITIES OF Markhamia lated compound, using the Prism 3.0 software package (GraphPad Software, La Jolla, CA). For this, the assay data were fitted, by non-linear regression, to the variable-slope sigmoidal dose–response formula y5100/ (1z10(logIC502x)H), where H is the Hill coefficient or slope factor (Singh and Rosenthal, 2001). Selectivity indexes (SI) were calculated, from the results of the assays of antimalarial and cytotoxic activities, as (IC50 for the L-6 cells)/(IC50 for a strain of P. falciparum). The values given in the Table are mean results (and S.D.) for either two independent assays (extracts) or three (pure compounds), with each assay run in duplicate. 395 ethyl-acetate extract had the best antimalarial activity, with similarly low IC50 and similarly high SI (.50) recorded for the two strains of P. falciparum that were tested (see Table). Fractionation of the ethyl-acetate extract by flash and successive column chromatography yielded eight compounds. Their structures were established, by 1H- and, 13 C-NMR spectroscopy (including one- and two-dimensional techniques) and mass spectrometry, as 2-acetylnaphtho[2,3-b]furan4,9-dione (1; Rao and Kingston, 1982), 2-acetyl-6-methoxynaphtho[2,3-b]furan-4,9dione (2; Zani et al., 1991), oleanolic acid (3; Mahato and Kundu, 1994), pomolic acid (4; Mahato and Kundu, 1994), 3-acetylpomolic acid (5; Mahato and Kundu, 1994), tormentic acid (6; Delgado et al., 1989; Mahato and Kundu, 1994; Li et al., 2009), b-sitosterol (7; Kovganko et al., 1999) and b-sitosterol-3O-b-D-glucopyranose (8; Moghaddam et al., 2007) (Fig. 1). Compounds 1, 2, 4 and 5 were then evaluated for their activities against P. falciparum, L. donovani and T. b. rhodesiense, to check whether or not each could contribute to the revealed antiprotozoal activity of the crude ethyl-acetate extract [the anti- RESULTS AND DISCUSSION In the present study, three extracts of the stem bark of M. tomentosa were evaluated in vitro for their activities against (the chloroquine-resistant K1 and W2 strains of) P. falciparum, L. donovani, and T. brucei rhodesiense — parasites that can cause human malaria, visceral leishmaniasis and African trypanosomiasis, respectively. The TABLE. In-vitro antiprotozoal and cytotoxic activities of the three crude extracts and isolated compounds Mean (S.D.) median inhibitory concentration (mg/ml): Plasmodium falciparum (K1) Sample P. falciparum (W2) Leishmania donovani Trypanosoma brucei rhodesiense Selectivity index L-6 cell line P. P. falciparum falciparum (K1) (W2) CRUDE EXTRACT Hexane Ethyl-acetate Methanolic .5 4.93 (0.39) .5 2.81 (0.06) 1.46 (0.12) .5 .5 ND .5 .5 1.5 .5 83 .90 .90 18 .50 – 17 .50 – ISOLATED COMPOUND 1 2 4 5 0.11 0.44 3.47 2.10 (0.02) 0.16 (0.02) (0.04) 0.93 (0.04) (0.90) .5 (0.7) .5 0.10 (0.02) 0.016 (0.004) 0.77 (0.04) 0.05 (0.01) 0.31 (0.03) .5 3.40 (0.7) .5 0.1 0.1 4.2 16.3 REFERENCE DRUG Chloroquine Miltefosine Melarsoprol Podophyllotoxin ND, Not determined. 0.094 (0.042) 0.039 (0.002) 0.12 (0.05) 0.003 (0.002) 0.007 0.9 0.2 1.2 7.8 0.6 0.1 13.7 – 396 TANTANGMO ET AL. FIG. 1. Structures of the isolated compounds 1–6. protozoal and cytotoxic activities of compounds 3 and 6 had already been investigated by Ngangoue (2008)]. Compounds 1 and 2 exhibited strong activities not only against the two strains of P. falciparum but also against the leishmanial amastigotes and trypanosomes tested (0.01 mg/ml,IC50,0.9 mg/ml; see Table). Unfortunately, both compounds also showed strong cytotoxic activity when tested against the L-6 cell line (IC5050.1 mg/ ml). Compounds 4 and 5 showed weak antiprotozoal activities and relatively low SI (see Table). Both active compounds, 1 and 2, possess an anthraquinone skeleton and are derived from lapachol (Fig. 2), a compound that is known to have antiparasitic activities (Hussain et al., 2007). Compared with 1, 2 bears an additional methoxy group at C-6, which may explain the differences in the activities of the two compounds. Several naphthoquinones and anthraquinones from plant sources, and synthetic derivatives, have been reported to possess good antimalarial and other antiprotozoal activities in vitro (Ribeiro-Rodrigues et al., 1995; Sittie et al., 1999; Onegi et al., 2002; Mbwambo et al., 2004; Noungoue et al., 2009). Furthermore, the naphthoquinone sterekunthal A (Fig. 2), isolated from the root bark of Stereospermum kunthiamum (which, like M. tomentosa, is a member of the Bignoniaceae), was found to exhibit good activity in vitro against P. falciparum, with an IC50 of 1.3 mg/ml (Onegi et al., 2002). Thus, the good antimalarial activity of the ethylacetate extract of M. tomentosa could be ANTIPROTOZOAL ACTIVITIES OF Markhamia 397 FIG. 2. Structures of lapachol and sterokunthal A. explained, at least partially, by the occurrence of the two naphthoquinones 1 and 2. Unfortunately, the antimalarial activity of these two compounds may reflect general toxicity, as shown by the low IC50 recorded against the mammalian cell line. The crude ethyl-acetate extract, which gave a much better SI for its antimalarial activity than any of the isolated compounds, must contain other compounds that moderate the antimammalian cytotoxicity of these two naphthoquinones. The extract may, of course, also contain antimalarial compounds that were not isolated in the present study. The present results highlight the antimalarial potency of naphthoquinones derivatives and partially validate the use of M. tomentosa, in Cameroonian folk medicine, to cure malaria. The authors thank the European Commission for a Marie Curie Post-doctoral Fellowship (awarded to B.N.L.) and the Academy of Sciences for the Developing World (TWAS) for awarding a research grant (07-141 LDC/CHE/AF/ AC-UNESCO FR: 3240171776) to the University of Yaoundé I’s TWAS Research Unit. The assistance of M. Nana (of the National Herbarium of Cameroon), in the collection and the identification of plant material, is gratefully acknowledged. ACKNOWLEDGEMENTS. REFERENCES Adesanya, S. A. & Nia, R. (1997). Palustrine from Markhamia tomentosa. Nigerian Journal of Natural Products and Medicine, 1, 39–40. Adjanohoun, E. J., Aboubakar, N., Dramane, K., Ebat, M. E., Ekpere, J. E., Enow-orock, E. 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