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anthoxylum chiloperone leaves extract: First sustainable Chagas diseasereatment
aria Elena Ferreiraa,1 , Gerardo Cebrián-Torrejónb,1 , Alba Segovia Corralesa , Ninfa Vera de Bilbaoa ,iriam Rolónc , Celeste Vega Gomezc , Karine Leblancb , Gloria Yalufa , Alicia Schininia , Susana Torresa ,
lva Sernaa, Antonieta Rojas de Ariasc, Erwan Pouponb, Alain Fournetd,∗
Department of Tropical Medicine, Casilla de Correo 2511, Instituto de Investigaciones en Ciencias de la Salud Asunción, Universidad Nacional de Asunción, ParaguayLaboratoire de Pharmacognosie, UMR 8076 CNRS, Faculté de Pharmacie, Université Paris-Sud, rue Jean-Baptiste Clément, 92296 Châtenay-Malabry Cedex, FranceCentro para el Desarrollo de la Investigación Científica (CEDIC/FMB/Diaz Gill Medicina Laboratorial), Asunción, ParaguayIRD UMR217, Laboratoire de Pharmacognosie, Faculté de Pharmacie, rue Jean-Baptiste Clément, 92296 Châtenay-Malabry, France
r t i c l e i n f o
rticle history:eceived 26 August 2010eceived in revised form 8 November 2010ccepted 11 November 2010vailable online 4 December 2010
Ethnopharmacological relevance: Zanthoxylum chiloperone var. angustifolium Engl. (Rutaceae) stem barkis used traditionally in Paraguay for its antiparasitic properties. Canthin-6-one is main compound iso-lated from Zanthoxylum chiloperone var angustifolium with broad spectrum antifungal, leishmanicidal andtrypanocidal activities.Aim of the study: The qualitative and quantitative characterization and the isolation of main alkaloidalcomponents of different organs of Zanthoxylum chiloperone are investigated by HPLC–UV–MS. The invitro biological activity of each extract against trypomastigote and amastigote forms of Trypanosomacruzi parasites were evaluated, then comparison the in vivo efficacy of the ethanolic leaves extract ofZanthoxylum chiloperone with reference drug, benznidazole, in acute Trypanosoma cruzi infected micewhen administered by oral route. We have also evaluated the mutagenic and cytotoxic activity of the maincomponent of Zanthoxylum chiloperone, i.e. canthin-6-one, by mouse bone marrow micronucleus test.Materials and methods: The compositions of the ethanol extracts obtained after the maceration processwere studied by HPLC–UV–MS methods. The quantitation analysis was performed by external standardmethod, using a calibration curve constructed utilizing solutions containing different concentrationsof the reference samples. The anti-trypomastigote activity was evaluated by the lysis effect on mouseblood trypomastigotes (Y strain Trypanosoma cruzi). The anti-amastigote Trypanosoma cruzi activity wasevaluated by a modified colorimetric method with chlorophenol red-�-d-galactopyranoside (CPRG). Thecytotoxicity of extracts and compounds was performed on NCTC 929 cells. The in vivo efficacy of theethanolic leaves extract of Zanthoxylum chiloperone and benznidazole, in acute Trypanosoma cruzi (twodifferent strains) was evaluated in Trypanosoma cruzi infected mice; the drugs were administered by oralroute. The mortality rates were recorded and parasitaemias in control and treated mice were determinedonce weekly for 70 days. The mutagenic and cytotoxic activity of the main component of Zanthoxylumchiloperone, canthin-6-one, by mouse bone marrow micronucleus test.Results: Canthin-6-one was the main compound of stem and root bark and 5-methoxy-canthin-6-one inleaves and fruits. The ethanolic leaves extract, canthin-6-one and benznidazole presented, approximately,the same level of in vitro activity against trypomastigote and amastigote forms of Trypanosoma cruzi. Wehave also evaluated the mutagenic and cytotoxic effects of canthin-6-one by micronucleus test in mice.This test showed any mutagenic and cytotoxic damages. The effects of oral or subcutaneous treatments at10 mg/kg daily for 2 weeks with the ethanolic extract of leaves of Zanthoxylum chiloperone were examined
in Balb/c mice infected acutely with Trypanosoma cruzi (CL or Y strain) and compared with benznidazole at
hese e
50 mg/kg for 2 weeks. In t were significantly reduced witConclusions: This study havereducing Trypanosoma cruzi pacommunities of Paraguay becendemic infection, Chagas dise
xperiments, 70 days after infection, parasitaemia and serological response
h the oral ethanolic extract treatment compared with reference drug.shown the efficacy of the leaves extract of Zanthoxylum chiloperone inrasitaemia in vivo assays and could be welcomed by scientific and rural
ause it could help them towards the use of local resources to treat anase, affecting 20% of the population of this country.
Currently available treatment of Chagas disease on the markets limited to only two drugs: nifurtimox (Lampit®, Bayer) and ben-nidazole (Lafepe®, Brazil). These drugs present severe side effectsnd are most successfully used in the acute infection and earlyhronic diseases (de Andrade et al., 1996). Generally, the diseases diagnosed in the chronic phase, when the therapy becomes inef-cient (Cerecetto and Gonzalez, 2002; Caldas et al., 2008). There
s an urgent need for new drugs for the chemotherapy of Chagasisease (Rassi et al., 2010). Although new and efficient treatmentsgainst Chagas disease are urgently needed, only one class of drugss in clinical development, i.e. triazoles, which have emerged as newotential treatments (Ribeiro et al., 2009).
Recently, we have reported the trypanocidal effect in experi-ental Chagas disease of canthin-6-one and some of its analogs
solated from a plant collected in Paraguay, Zanthoxylum chiloper-ne var. angustifolium Mart. Engl. (Rutaceae) (Ferreira et al., 2002b,007). In that study, the results indicated that canthin-6-one exhib-
ted trypanocidal activity in vivo in the mouse model of acuter chronic infection. In addition, the total extract of alkaloidstotum) of Zanthoxylum chiloperone stem bark led to high lev-ls of parasitological clearance. In this work, we had chosen anmpirical approach associating the in vitro biological tests againstarasites and activity-guided fractionation, mainly because nothnopharmacological approach was possible to guide the selec-ion of active plants against Chagas disease due to the absence ofxternal symptoms, but this plant is used to treat parasitic diseases.fter various chemical and biological studies with canthin-6-one
ype compounds (Thouvenel et al., 2003; Soriano-Agaton et al.,005; Lagoutte et al., 2008) and the establishment of an agree-ent between IRD (Institut de Recherche pour le Développement)
nd the Drugs for Neglected Diseases initiative (DNDi), a non-rofit product development partnership, we have entered intowo synergistic agreements to identify and develop new promis-ng drug candidates against Chagas disease. This collaboration hasllowed the optimization and development of canthin-6-one alka-oids. These molecules are the subject of patent owned by a jointooperation between IRD and the National University of AsunciónUNA) in Paraguay (Ferreira et al., 2002a). In the present study, weish to disclose a new approach to develop a treatment of Cha-
as disease from Zanthoxylum chiloperone, demonstrating that anxtract prepared from Zanthoxylum chiloperone leaves holded theame activity as reference drug, benznidazole and canthin-6-one.
The aims of this study are (i) the qualitative and quantitativevaluation by HPLC–UV–MS methods of the content of active com-ounds, namely canthin-6-one and its close analogs in differentrgans of the plant (leaves, stem bark, roots bark, resin and fruits),ii) the in vitro biological activity of each extract, against trypo-
astigote and amastigote forms of Trypanosoma cruzi parasites andiii) finally comparison of the in vivo efficacy of the ethanolic leavesxtract of Zanthoxylum chiloperone with reference drug, benznida-ole, in acute Trypanosoma cruzi infected mice when administeredy oral route. We have also evaluated the mutagenic and cyto-oxic activity of the main component of Zanthoxylum chiloperone,anthin-6-one, by mouse bone marrow micronucleus test (Heddle,973).
. Materials and methods
.1. General experimental procedures
Yields refer to chromatographically and spectroscopicallyomogeneous materials, unless otherwise stated. Extraction wasonitored by TLC carried out on Merck Kieselgel silica gel plates
rmacology 133 (2011) 986–993 987
(60F-254) using UV light as visualizing agent and sulfuric vanillin orDragendorff reagent and heat as developing agent. Merck Kieselgelsilica gel (60, particle size 40–63 �m) was used for flash chro-matography. NMR spectra were recorded on an AM-400 Brukerspectrometer, calibrated using undeuterated solvent as an internalreference. IR spectra were recorded on Vector 22 Bruker spectrom-eter and values are reported in cm−1 units. Mass spectra wererecorded on a Bruker Esquire-LC mass spectrometer. An ion trapmass spectrometer with Electrospray (ESI) and Atmospheric Pres-sure Chemical (APCI) Ionization source was employed at “Serviced’Analyse des Médicaments et Métabolites”, SAMM, Université Paris-Sud, Châtenay-Malabry, France.
2.2. Plant material
Leaves, stem bark, roots, fruits and resin of Zanthoxylum chiloper-one var. angustifolium Engl. were collected by Maria Elena Ferreira,in Paraguay near Piribebuy, Department of Cordillera and identifiedby N. Soria (Department of Botany, National University of Asuncion,Paraguay) (Spichiger and Stutz de Ortega, 1987). A voucher spec-imen (MEF 55) has been deposited at the Herbarium of ChemicalSciences Faculty, San Lorenzo, Paraguay.
2.3. Extraction and isolation
Open air-sun drying was carried out with the different parts ofthe plant. Powdered dried stem bark of Zanthoxylum chiloperonevar. angustifolium (1.3 kg) was basified with ammonia, extractedin a soxhlet apparatus with CH2Cl2 and MeOH separately andsuccessively for 3 days to afford 23 g and 30 g of crude extractsrespectively.
Canthin-6-one 1, 5-methoxycanthin-6-one 2 and canthin-6-one N-oxide 3 were isolated as previously described (Thouvenelet al., 2003). Physical and spectral data were used to determine thechemical structure of the compounds and compared to referencesamples and literature data. Benznidazole (N-benzyl-1,2-nitro-1-imidazole-acetamide) was purchased from Roche, Buenos Aires,Argentina, and used as a reference drug.
Powdered dried leaves of Zanthoxylum chiloperone var. angusti-folium (500 g) were basified with ammonia, extracted in a Soxhletapparatus separately and successively with CH2Cl2 and MeOH for 3days to afford 10 g and 13 g respectively of crude extracts. The yieldsof the dichloromethane and methanolic extracts were approxi-mately 2% and 2.6% respectively of the plant powder. The CH2Cl2extract was subjected to silica gel flash column chromatography,eluted with cyclohexane/ethyl acetate (5:5), to obtain 42 fractions.Fractions 20–35 yielded 2 (105 mg) as a pure compound (1% and0.02% respectively from the dichloromethane extract and plantpowder).
To evaluate the chemical content of the different parts of theplant we have worked with powdered dried leaves, stem bark,fruits, roots and resin (2 g, 5 g, 24 g, 30 g and 400 mg). All specimenswere basified with ammonia, extracted by ethanol maceration dur-ing 2 days, changing the solvent three times to afford 150 mg,115 mg, 1 g, 6 g, 20 mg respectively of crude ethanol extracts.The yields of the alcohol extracts were approximately 7.5%, 2.3%,4.1%, 20% and 5% respectively. The compositions of the ethanolextracts obtained after the maceration process were studied byHPLC–UV–MS methods.
2.4. Analysis by HPLC–UV–MS methods
2.4.1. HPLC–UV–MS qualitative studyHPLC analyses of natural products is usually performed using a
large set of standards, and the measurements are typically achievein the gradient elution mode, whereby the retention time depends
Fig. 1. HPLC–UV–MS chromatogram of reference compounds.
n a complicated manner on the thermodynamic characteristics ofetention (Carini et al., 1998; Nuengchamnong et al., 2009).
A LC/MSD 1100 series + 1100 series (AGILENT TECHNOLOGIES)pectrometer was utilized in the HPLC study of the extracts using150 mm × 4, 6 mm stainless-steel column Sunfire (WATERS) C18.he eluents were (A) H2O + TFA 0.1% and (B) MeOH + 0.1% TFA. Sep-rations were performed at room temperature by solvent gradientlution from 50% A/50% B to 20% A/80% B in 20 min then to 100% Bn 12 min at a flow rate of 1 mL/min.
The UV measurement was made at 240 nm and 260 nm. Thenjection volume was 5 �L. Peak identification was performed byomparison of the retention time and UV absorption and masstudy at the different wavelengths with the compounds canthin--one 1, 5-methoxy-canthin-6-one 2 and canthin-6-one N-oxide 3s references (Fig. 1).
The Fig. 2 illustrates the HPLC–UV chromatograms of the ethanolnd dichloromethane extracts of different parts of the plant Zan-hoxylum chiloperone.
From the qualitative point of view, we considered the peaks ofhe three canthinones (1–3) (Fig. 1) as references and we searchedor these reference peaks in the HPLC–UV chromatograms of theifferent extracts (Fig. 2).
.4.2. HPLC–UV quantitative studyThe quantitation analysis was performed by external standard
ethod, using a calibration curve constructed utilizing solu-ions containing different concentrations of the three referenceamples (1–3). The stock solutions were prepared in a mixtureeOH + DMSO, where these betacarboline type alkaloids dissolveuch better than in methanol, and then diluted with the mobile
hase in various proportions.The proposed method was used for determining the content
n betacarboline type alkaloids in the different extracts preparedrom plant materials sometimes kept in ethanol with stirring, andometimes extracted by soxhlet reflux with dichloromethane as anxtractive solvent. All the samples for HPLC analysis were operatedirectly without any previous purification.
A modular system comprised of 600 E controller + pump and
2996 PDA UV (WATERS) detector was utilized in the HPLC–UV
00 �g/mL were utilized. Each determination was carried out inriplicate.
rmacology 133 (2011) 986–993
The calibration curves showed the linearity of the detector overthe tested range (10–300 �g/mL). The respective coefficients ofdetermination (R2) showed a value of 0.999, for ease of reference,Supplementary Table 1 illustrates the corresponding equations ofthe respective curves. The quantitative evaluation of the peaks pre-viously identified in the crude extracts was made by integration ofthe peak area and comparison with the area of the correspondingreference peak.
2.5. Biological assays
2.5.1. Anti-trypomastigote activityThe lysis effect on mouse blood trypomastigotes (Y strain) was
presented (Table 2) for extracts of Zanthoxylum chiloperone andcanthin-6-one type compounds at 250 �g/mL (Chiari et al., 1996).Gentian violet was used as the reference drug.
2.5.2. Anti-amastigote activityThe activity was evaluated with chlorophenol red-�-d-
galactopyranoside (CPRG) by a modified colorimetric methoddescribed previously (Buckner et al., 1996; Rolon et al., 2006).
2.5.3. Cytotoxic activityThe bioassay was performed as previously described by Rolon
(Rolon et al., 2006). Briefly, NCTC 929 cells were plated in 96-wellmicrotitre plates at 3 × 104 cells/well in 100 �L of growth medium.The cells were grown overnight at 37 ◦C in 5% CO2. Thereafter, themedium was removed and the compounds were added in 200 �L ofmedium for 24 h. After incubation, 20 �L of 2 mM resazurin solutionwas added to each well. The plates were incubated for 3 h to allowoptimal oxidation–reduction. The plates were read at 490 nm and595 nm on a microplate reader (Bio-Tek Instruments Inc., Winooski,VT, USA).
2.6. In vivo studies
2.6.1. Mice and parasitesFemale and male BALB/c mice were bred at the “Instituto de
Investigaciones en Ciencias de la Salud” IICS (Asunción, Paraguay)and were 6–8 weeks of age when used. In this experiment, theclone CL of Trypanosoma cruzi supplied by Dr. B. Zingales, Sao Paulo,Brazil was used (Cano et al., 1995). Routine maintenance of the Try-panosoma cruzi strain is carried out in BALB/c mice inoculated byintraperitoneal route every 14 days.
All mice were infected intraperitoneally with 5000 blood try-pomastigotes of CL strain or 100 blood trypomastigotes of Y strainfrom blood. We carried out in acute infection two separate experi-ments to evaluate the in vivo efficacy of drugs. The treatments werestarted 7 days after inoculation of the parasites. The mice wererandomly divided into groups between 8 and 12 mice. A referencedrug, benznidazole (Roche) was selected for all experiments. Ben-znidazole and the rest of drugs were diluted in 50 �L of phosphatebuffered saline (PBS). Benznidazole was administered to BALB/cmice by the oral route in regimens of 50 mg/kg daily for 2 weeks.The ethanolic extract of Zanthoxylum chiloperone was administeredby oral or subcutaneous route at 10 mg/kg for 2 weeks.
2.6.2. Treatment outcomeThe mortality rates were recorded. Parasitaemias in control and
treated mice were determined once weekly for 70 days in tail-vein
blood and the mortality rate was recorded.
Sera from acute infected mice were tested once by ELISA(Enzyme Linked Immuno Sorbent Assay) 70 days post infection.A locally produced ELISA kit (Chagas test, IICS, Asunción, Paraguay)was used following the procedure recommended by the manufac-
M.E. Ferreira et al. / Journal of Ethnopharmacology 133 (2011) 986–993 989
anoli
tr
2
ag
Fig. 2. HPLC–UV chromatogram of eth
urer. The optical density values were obtained using an ELISA plateeader (Titerek Uniscan I).
.6.3. Mutagenicity study (micronucleus test)Five-animal groups were co-treated i.p. twice with 6.25, 12.5
nd 25 mg/kg of canthin-6-one. Negative and solvent-controlroups were also included. For experiments, after the treatments
c extracts of Zanthoxylum chiloperone.
(24 h), femurs were exposed and sectioned opened and the bonemarrow was gently flushed out using fetal calf serum. After cen-
trifugation (300 × g, 5 min) the bone marrow cells were smearedon glass slides, coded for blind analysis, air-dried and fixed in abso-lute methanol for 5 min. The smears were stained with Giemsafor detecting micronucleated polychromatic erythrocytes (MNPCE)(Schimid, 1975). For each animal, three slides were prepared and
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000 polycromatic erythrocytes (PCE) were counted to determinehe frequency of MNPCE. To determine the cytotoxicity of this plant,000 normocromatic erythrocytes (NCE) were counted, as well ashe frequency of PCE within the same microscope fields. Then, theCE/NCE ratio was calculated. The slides were analyzed in OlympusH-2 microcopy (10 × 100).
.7. Statistical analysis of data
The means and standard deviations were calculated and the dif-erences between groups were determined using Student’s t-testr the Kruskal–Wallis nonparametric analysis of variance test foromparing two groups. Significance was established for a P valuef <0.05.
In order to analyze the antimutagenic activity of canthin-6-ne, the frequency of the MNPCE from the treated groups wereompared to the results obtained from the positive control groupscyclophosphamide) by Student’s t-test. The same results were alsoompared to the negative control group (water) by the t-test. Palues less than 0.05 were considered as indicative of significance.
. Results
.1. Occurrence of canthin-6-one alkaloids in each organ ofanthoxylum chiloperone
The composition of each organ of Zanthoxylum chiloperoneas analyzed by HPLC–UV–MS study of the ethanol extracts
f stem bark, leaves, fruits, root bark and resin as well as theichloromethane extracts of stem bark and leaves. The characteri-ation of the extracts was studied merely from the analytical pointf view.
The qualitative study identified the main compounds inanthoxylum chiloperone, such as canthin-6-one alkaloids, canthin--one 1, 5-methoxy-canthin-6-one 2 and canthin-6-one N-oxide. Canthinone compounds were identified by their retention timestR) and by their mass spectra respectively. The chromatographicrofiles of the reference compounds are presented in Fig. 1 andhe profiles of the respective ethanol extracts are presented inig. 2. Canthin-6-one 1 was identified as the major compound inhe stem bark and root bark, while 5-methoxy-canthin-6-one 2 washe major compound in the leaves, fruits and resin, lastly canthin--one N-oxide 3 was present in the resin and in the stem bark (seeable 1).
The quantitative analysis was performed by external standardethod, using a calibration curve constructed utilizing solutions
ontaining different concentrations of the three reference sam-les (see Supplementary Table 1). The results of the quantitativeest filled out the canthinone qualitative analysis of the differentxtracts and helped us to determine the real concentration of eacholecule in the extracts (Table 1).The concentrations of canthin-6-one 1 in the ethanol extract
f the stem bark and the root bark are 64.7 and 8.2 mg/g extractespectively, while the concentrations of 5-methoxycanthin-6-onein the ethanol extract of the leaves, the fruits and the resin are
0.8, 3.5 and 5 mg/g extracts respectively, finally the concentrationsf canthin-6-one N-oxide into the ethanol extracts of the stem barknd resin are 8.2 and 1.9 mg/g of extract respectively (Table 1).
.2. In vitro biological activity of canthin-6-one compounds
owards Trypanosoma cruzi
In order to evaluate the trypanocidal activity, we studied the initro activity of each extract prepared from the different organs ofhe plant against the trypomastigote forms of Trypanosoma cruzi
rmacology 133 (2011) 986–993
(Y strain), the etiologic agent of Chagas disease. Thus, the ethano-lic leaves extract, canthin-6-one 1 and 5-methoxy-canthin-6-one 2lysed by 75–79% of parasites and the standard drug benznidazole by87% (see Table 2). To evaluate anti-amastigote activity, amastigotesproliferating in fibroblasts were used. The results are presented inTable 2, canthin-6-one 1 and 5-methoxy-canthin-6-one 2 at con-centration of 15.1 �M showed the highest levels of activity (%AA90.0 and 66.4 respectively) after 7 days of incubation. Their activ-ities were comparable with that of benznidazole, although at aconcentration five-fold lower.
The cytotoxicity of the different extracts and compoundswere also evaluated using fibroblasts cell culture (NTC 929) atconcentration of 15.1 �M, which canthin-6-one 1 and 5-methoxy-canthin-6-one 2 showed the highest anti-amastigote activity. Nosignificant toxicity was observed at that concentration in fibroblastcells (Table 2).
3.3. In vivo biological activity of canthin-6-one compoundstowards Trypanosoma cruzi
The ethanolic leaves extract of Zanthoxylum chiloperone waswell tolerated by the mice and we have not observed secondaryside effects in any of the experiments. BALB/c mice were infectedintraperitoneally with 5000 trypomastigotes of CL strain or with100 trypomastigotes of Y strain. During the infection with CL strain,the mean value of parasitaemia obtained at the 22nd day post-infection was 7.7 ± 1.4 × 105 parasites per mL blood in control, 1.2and 3.0 × 105 in benznidazole and oral ethanol extract-treated mice(Fig. 3A). The parasitaemia at day 50 for the benznidazole andethanol extract-treated mice was significantly reduced (P < 0.01)compared to the control group. The efficacy of these treatments wasconfirmed by the absence of parasites and a significant reductionof serological response at 60 days post treatment when com-pared with benznidazole and control groups (see Table 3). In thecase of infection with Y strain, the mean value of maximum par-asitaemia was obtained at the 19th day post-infection for thecontrols (2.5 × 104) and the 12th for the benznidazole and ethano-lic extract-treated mice, 1.9 and 1.8 × 104 respectively. For thesegroups, negative parasitaemia were observed 1 week later. Themean parasitaemia at day 28th for benznidazole and oral canthin-6-one treated mice were significantly reduced (P < 0.0001 versuscontrols). All untreated mice infected with CL strain presented pos-itive parasitaemia after 60 days of experiment (Fig. 3A), on theother hand treated mice with benznidazole or ethanol extract hadnegative parasitaemia (see Table 3). In the experiment with Y Try-panosoma cruzi strain infection, the percentage of mortality wasmore important for untreated mice (60%) compared to treatedgroups (Fig. 3B). In the treated mice, the absence of parasites inblood was attested by control of parasitaemia 33 days after infec-tion, and a significant decreased (P < 0.05) of ELISA serology 30 dayspost-infection was observed, no significant differences were notedwhen serology was evaluated at 60 days post infection (see Table 3).During all experiments we have observed no apparent side effectresulting from treatment with the ethanol extract of Zanthoxylumchiloperone.
3.4. Mutagenicity study (micronucleus test)
The results obtained from the mouse bone marrow cells after24 h of administration with ethanolic canthin-6-one are presentedin Table 4. The frequency of micronucleated polychromatic ery-
throcytes (MNPCE) means (per 1000 PCE) in the mice were 8, 12and 6 in the groups treated with ethanolic canthin-6-one at 6.25,12.5 and 25 mg/kg body weight respectively, while that of the pos-itive control (cyclophosphamide) was 106. For all doses the resultsshowed a significant reduction of MNPCE frequencies compared
M.E. Ferreira et al. / Journal of Ethnopharmacology 133 (2011) 986–993 991
Table 1Concentrations of active compounds in the various extracts prepared from different organs of Zanthoxylum chiloperone.
Table 2In vitro trypanocidal activity of Zanthoxylum chiloperone extracts (trypomastigote and amastigote forms) and cytotoxic effects on Fibroblast cell line.
Stem bark CH2Cl2 51 – NT NDEtOH 56 – NT NDLeaves CH2Cl2 73 – NT NDEtOH 78 – NT NDFruits EtOH 45 – NT NDRoot bark CH2Cl2 48 NT NDCanthin-6-one 79 15.1 90.0 ND5-Methoxy-canthin-6-one 75 15.1 66.4 NDGentian violet 100 – NT NDBenznidazole 87 192 97.5 ND
ND = not detected at 15 �M; NT = not tested.a % Lysis = percentage of parasite lysis at 250 �g/mL of the compound.b Results are the mean of the three independent experiments with a SD less than 10% in all cases.c AA, percentage of anti-amastigote activity.
Fig. 3. Effects of benznidazole at 50 mg/kg and ethanolic extract of Zanthoxylum chiloperone at 10 mg/kg on parasitaemia in BALB/c mice with acute Trypanosoma cruziinfection by CL strain (A) or by Y strain (% mortality) (B). HPLC–UV–MS chromatogram of ethanolic extracts of Zanthoxylum chiloperone and effects on parasitaemia in BALB/cmice with acute Trypanosoma cruzi infection by Y strain (% mortality).
992 M.E. Ferreira et al. / Journal of Ethnopharmacology 133 (2011) 986–993
Table 3Effect of benznidazole, and ethanolic Zanthoxylum chiloperone leaves extract in Trypanosoma cruzi infected mice (CL or Y strain) as judged by the absence of parasitaemia andserological response (Test ELISA).
a P < 0.05 versus control.b P < 0.02 versus control.c P < 0.05 versus benznidazole.d P < 0.001 versus control.
o the positive control group (P < 0.05). The negative and solventontrol groups were all statistically different compared to positiveontrol group (P < 0.05).
. Discussion
The present study was conducted in order to evaluate thehemical composition of each organ of Zanthoxylum chiloperonen canthin-6-one type compounds previously identified as try-anocidal compounds (Ferreira et al., 2007). The trypanocidal,ytotoxic and antimutagenic activities of the ethanolic extract ofhis plant have been evaluated. We have focused this study on
he ethanolic extract of Zanthoxylum chiloperone leaves becausesing the leaves we can protect the natural resource, this resource
s renewable, easy to prepare and can be produced in impor-ant scale by local rural communities and could have implicationsor local sustainable development. For this, we have established
able 4requencies of micronucleated polychromatic erythrocytes in bone marrow of mice treat
Treatment Number of animalsa
Water (negativecontrol)
5
Water + HCl (negativecontrol)
5
Cyclophosphamide(positive control)
5
Canthin-6-one6.25 mg/kg 512.5 mg/kg 525 mg/kg 5
a 1000 polychromatic erythrocytes per mouse; statistically different from the positive
an analytic method (HPLC) to control the quality of Zanthoxy-lum chiloperone and the trypanocidal activity at each step ofproduction.
Current treatment of Chagas disease is limited to only twodrugs: benznidazole (Lafepe-benznidazole®, Brazil) and nifurtimox(Lampit®, Bayer). These drugs are restricted to the acute phase andin early chronic disease and the children treatment. The geneticdiversity of the parasite Trypanosoma cruzi was demonstrated(Villareal et al., 2004) and various authors have reported impor-tant variations in susceptibility to drugs (Veloso et al., 2001). Inthis study we have shown that the leaves extract of Zanthoxylumchiloperone was active in vivo against two different strains of Try-
panosoma cruzi.
By our study, we have chosen a new approach, the developmentof a sustainable treatment from ethnopharmacological (Thouvenelet al., 2003; Ferreira et al., 2007), phytochemical (Soriano-Agatonet al., 2005) and pharmacological (Ferreira et al., 2002a,b; Lagoutte
ed with canthin-6-one or cyclophosphamide.
Micronucleated polychromaticerythrocytes (MPE)
No. % ± SD
10 0.2 ± 0.02a
3 0.060 ± 0.005a
106 2.12 ± 0.10
8 0.16 ± 0.02a
12 0.24 ± 0.03a
6 0.12 ± 0.01a
control (cyclophosphamide) P < 0.05.
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M.E. Ferreira et al. / Journal of Eth
t al., 2008) results. We have identified and evaluated the yield ofhe main components of each organ of Zanthoxylum chiloperone,anthin-6-one 1, 5-methoxy-canthin-6-one 2 and canthin-6-one-oxide 3. Separately, these molecules had previously demon-trated their efficacy on experimental Chagas disease (Ferreirat al., 2007).
Previous studies (Thouvenel et al., 2003; Lagoutte et al., 2008),ad shown an interesting antifungal activity of canthin-6-one,ut its mechanism of action, albeit unclear at this time, was dif-erent to known antifungal drugs (Molina et al., 2000; Urbina,002; Urbina et al., 2003a,b), generally related to the ergosteroliosynthesis.
In conclusion, this study could be welcomed by scientific andural communities of Paraguay because it could help them towardshe use of local resources to treat an endemic infection, Chagas dis-ase, affecting 20% of the population of this country (WHO, 2002).his work has shown that the collaboration between ethnophar-acologists, parasitologists, chemists, and physicians can open
erspectives to develop a new drug from ethnopharmacologicalnformation.
cknowledgements
We thank the European Community for the financial supportMarie Curie Early Stage Training Fellowship of the European Com-
unity’s Sixth Framework Programme: contract BioMedChem, for.C.-T.) and A. Solgadi (SAMM) for MS experiments.
ppendix A. Supplementary data
Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.jep.2010.11.032.
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