ANTIPROTOZOAL ACTIVITIES OF COMPOUNDS ISOLATED FROM CROTON LOBATUS L

In a preliminary evaluation of ethnobotanically sel ected Beninese medicinal plants for their in vitro antiplasmodial activity, the methanolic extract of the aerial parts of C. lobatus was found to have significant activity against P. falciparum, antileishmainal and antiprotoazoal assays carried o ut on some of the isolated compounds. Phytochemical investigation of this extr act resulted in the isolation of five compounds: ti liroside (kaempferol-3O-β-D-(6-E-p-coumaroyl) glycopyranoside) ( 1), isovitexin (apigenin-6C-β-D-glucopyranoside) (2), vitexin (apigenin-8Cβ-D-glucopyranoside) ( 3), chlorogenic acid (acid-5O-cafféoylquinic) (4) and 4,5Odicaffeoylquinic acid ( 5). Vitexin and tiliroside showed the best in vitro antiplasmodial activity against P. falciparum, with IC50 values of 4.4 and 7.1 μM, respectively. Vitexin also exhibited strong in vitro leishmanicidal and antitrypanosomal activities agai nst Leishmania donovani amastigotes and Trypanosoma brucei rhodensiense trypomastigotes, with IC 50 values of 0.6 and 0.1 μM, respectively.


Introduction
Protozoal diseases, such as malaria, trypanosomiasis, and leishmaniasis, are a major threat for public health.Every year, malaria causes 300 to 500 million clinical cases and more than one million deaths, and there is increasing prevalence of malaria exhibiting resistance of Plasmodium falciparum to inexpensive, cheap standard treatments (Wellems and Plowe, 2001).In the same order of idea, leishmaniasis and trypanosomiasis are major causes of mortality and cause much economic hardship, particularly in the developing world.Unfortunately, there are few drugs available to treat these two diseases, and most of these drugs suffer from poor clinical efficacy and unwanted effects.There is an urgent need to discover new therapeutic agents for these parasitic diseases, and traditional medicine knowledge can be useful to open new ways in the field of antiprotozoal therapy.In a previous work, we reported in vitro antiprotozoal activity of Croton lobatus L. (Euphorbiaceae), a traditional plant used in Benin and in neighbouring country Togo, against malaria in folk medicine, towards both chloroquine-sensitive and chloroquine-resistant strains of P. falciparum (Adjanohoun et al., 1989;Weniger et al., 2004).

Plant material
Aerial parts of Croton lobatus L. was collected in January 2005 in the area of Weto, in the Atlantic region (Southern Benin).Botanical determination was performed by taxonomists from the Herbier National of Abomey-Calavi University in Benin, and voucher specimens (Hp 565a) were deposited at the same Herbarium.The aerial parts were air-dried, powdered (0.2 mm sieve), and subjected to extraction with methanol.

Extraction and isolation
Dry powdered aerial parts of C. lobatus (515g) were extracted with methanol using a Soxhlet extractor (Soxtec Avanti 2055 apparatus, Foss Tecator AB, Box 70, Se-263 21, Höganäs, Sweden).The dry methanolic extract (29.27g) dissolved in water and then partitioned with cyclohexane, methylene chloride and ethyl acetate.Each fraction was taken to dryness under vacuum and the residues were stored at room temperature.The ethyl acetate fraction (2.73g) was first chromatophied using Sephadex LH-20 exclusion chromatography (Sephadex LH20, 25 -100 µm, Merck), according to the method described by Houghton and Raman (1998).Two fractions (A, 855 mg and B, 962 mg) were obtained and purified using a combination of chromatographic methods such as high pressure liquid chromatography (HPLC), atmospheric column chromatography (CC) and preparative TLC chromatography.
Structural determination of the isolated compounds was carried out by spectrophotometric methods (1D and 2D NMR, mass and UV spectrometry).1D and 2D NMR spectrum were recorded at room temperature with a Bruker NMR spectrometer (200MHz and 300 MHz).

Biological assays Antiplasmodial activity
Quantitative assessment of in vitro antimalarial activity against the Kl resistant strain was determined by means of the microculture radioisotope technique based on the method previously described by Desjardins et al. (1979) and modified by Ridley et al. (1996).The assay uses the uptake of [ 3 H]hypoxanthine by parasites as an indicator of viability.Continuous in vitro cultures of asexual erythrocytic stages of Plasmodium falciparum were maintained following the methods of Trager and Jensen (1976).Compounds were tested against Kl strain (multidrug pyrimethamine/chloroquine resistant strain) (Thaithong and Beale, 1981).Initial concentration of each compound was 30 µg/ml diluted with two-fold dilutions to make seven concentrations, the lowest being 0.47 µg/ml.After 48 h incubation of the parasites with the compound at 37•C, [ 3 H]hypoxanthine (Amersham 115 Int., Buckinghamshire, UK) was added to each well and the incubation was continued for another 24 h at the same temperature.IC50 was calculated by linear interpolation between the two drug concentrations above and below 50% (Huber and Koella, 1993).Chloroquine and artemisinin were used as positive references.The values are means of two independent assays.Each assay was run in duplicate.

Antitrypanosomal and leishmanicidal assays Trypanosoma brucei rhodesiense
The assays were performed according to the procedures described by Freiburghaus et al. (1996).The compounds were dissolved in 10% DMSO, and working stock solutions of 1 mg/mL in serum containing culture medium were prepared.Diluted compounds (100 µL) were pipetted in duplicate into the first row of a 96-well microtiter plate (Costar, Corning, NY, USA).With complete culture medium, three-fold serial dilutions were prepared.After the addition of Trypanosoma brucei rhodesiense bloodstream-form trypanosomes from axenic culture, the concentrations of the compounds ranged from 500 to 0.07µg/mL.The total number of trypanosomes in each well was 2 × 102/100 µL.The plate was then incubated for 72 h at 37•C in 5% CO 2 .Two hours before the end of the incubation 10 µL of Alamar blue solution was added.Fluorescence was measured after 2 h of incubation with the dye Alamar blue in a fluorescence plate reader at 530 nm excitation and 590-nm emission wavelength (Cytofluor 2300, Millipore, Bedford, MA, USA) (Räz et al., 1997).IC50 values were calculated from the sigmoidal inhibition curve.The values are means of two independent assays.Each assay was run in duplicate.

Leishmanicidal activity
Fifty microliters 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% heat-inactivated FBS, was added to each well of a 96-well microtiter plate (Costar).Serial drug dilutions in duplicates were prepared covering a range from 30 to 0.041 µg/mL.Then, 105 axenically grown L. donovani amastigotes (strain MHOM/ET/67/L82) in 50 µL medium were added to each well and the plate incubated at 37•C under a 5% CO 2 atmosphere for 72 hrs.Ten microliters of resazurin solution (12.5 mg resazurin dissolved in 100mL distilled water) were then added to each well and incubation continued for a further 2-4 hrs.The plate was then read in a Spectramax Gemini XS microplate fluorometer (Molecular Devices, Sunnyvale, CA, USA) using an excitation wavelength of 536 nm and emission wavelength of 588 nm (Räz et al., 1997).Fluorescence development was measured and expressed as percentage of the control.Data were transferred into the graphic programe Softmax Pro (Molecular Devices), which calculated IC 50 values.The values are means of two independent assays.Each assay was run in duplicate.

Results and Discussion
In a previous work, the methanolic extract of aerial parts of C. lobatus was found to have significant activity against P. falciparum in vitro (Weniger et al., 2004).The phytochemical analysis of the active extract of aerial parts of C.lobatus led to the isolation and characterization of five compounds, identified as tiliroside , chlorogenic acid (acid-5-O-cafféoylquinic) (4) and 4,5-Odicaffeoylquinic acid (5) (Figure 1).All the data were consistent with the literature ( (Sala et al., 2003) and Platanus orientalis L. (Dimas et al., 2000).It showed a good antioxidizing property revealing using DPPH assay (IC 50 = 6µM) (Sala et al., 2003).It also showed a significant inhibition without cytotoxicity of the production of inflammatory intermediaries (NO, TNF alpha, IL-12, LPS, IFN) to murins macrophages.Isovitexin was isolated previously from Lythrum salicaria (Becker et al., 2005), Patrinia villosa (Peng et al., 2005), Thlaspi arvense (Pedras et al., 2003).It showed an antioxidative property (Pedras et al., 2003).Vitexin was isolated previously from the flowers and leaves of Ficaria verna (Tomczyk et al., 2002) and Terminalia catappa (Yun-Lian L. et al., 2000).(Kima et al., 2005).Dicaffeoylquinic acid activity concerns the inhibition of the growth of the tumoral HL-60 cells by induction of apoptose (Satoshi et al., 2005).All these compounds were evaluated for in vitro antiplasmodial activity to check whether or not they contribute to activity.As C. lobatus is also used to treat parasitic diseases in Togo folk medicine, the isolated compounds were also assayed for leishmanicidal and antitrypanosomal activities.IC 50 values are presented in Table 1.Three of the five compounds tested, vitexin (3), tiliroside (1) and 4,5-O-dicaffeoylquinic acid (5) showed mild antiplasmodial activity, with IC 50 values of 4.4, 7.1 and 9.7 µM, respectively.These results provide scientific evidence supporting the use of C. lobatus as an antimalarial remedy in folk medicine in Benin.Moreover, vitexin exhibited a prominent leishmanicidal effect with an IC 50 value comparable to that of the reference compound miltefosin, as well as significant antitrypanosomal activity against T. b. rhodensiense.These results provide scientific evidence supporting the use of C. lobatus to treat parasitic disease in traditional medicine in Togo.

Conclusion
The results obtained in this study showed the interest of the ethnopharmacological approach in the search for active substances against the parasitic pathology.The active tested compounds may, either to be models for the synthesis of more active or less toxic analogues, or to be used in association with antimalarial commercial drugs, as it was shown in recent works (Ramanitrahasimbola et al., 2006), as they may reverse the resistance of the parasite to antimalarial drugs.