Antitrypanosomal compounds from the essential oil and extracts of keetia leucantha leaves with inhibitor activity on trypanosoma brucei glyceraldehyde-3-phosphate dehydrogenase.
Keetia leucantha is a West African tree used in traditional medicine to treat several diseases among which parasitic infections. The dichloromethane extract of leaves was previously shown to possess growth-inhibitory activities on Plasmodium falciparum, Trypanosoma brucei brucei and Leishmania mexicana mexicana with low or no cytotoxicity (>100[micro]g/ml on human normal fibroblasts) (Bero et al. 2009,2011). In continuation of our investigations on the antitrypanosomal compounds from this dichloromethane extract, we analyzed by GC-FID and GC--MS the essential oil of its leaves obtained by hydrodistillation and the major triterpenic acids in this extract by LC--MS. Twenty-seven compounds were identified in the oil whose percentages were calculated using the normalization method. The essential oil, seven of its constituents and the three triterpenic acids were evaluated for their antitrypanosomal activity on Trypanosoma brucei brucei bloodstream forms (Tbb BSF) and procyclic forms (Tbb PF) to identify an activity on the glycolytic process of trypanosomes. The oil showed an [IC.sub.50] of 20.9 [micro]g/ml on Tbb BSF and no activity was observed on Tbb PF. The best antitrypanosomal activity was observed for ursolic acid with [IC.sub.50] of 2.5 and 6.5[micro]g/ml respectively on Tbb BSF and Tbb PF. The inhibitory activity on a glycolytic enzyme of T. brucei, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was also evaluated for betulinic acid, olenaolic acid, ursolic acid. phytol, [alpha]-ionone and [beta]-ionone. The three triterpenic acids and [beta]-ionone showed inhibitory activities on GAPDH with oleanolic acid being the most active with an inhibition of 72.63% at 20 [micro]g/ml. This paper reports for the first time the composition and antitrypanosomal activity of the essential oil of Keetia leucantha. Several of its constituents and three triterpenic acids present in the dichloromethane leaves extract showed a higher antitrypanosomal activity on bloodstream forms of Tbb as compared to procyclic forms, namely geranyl acetone, phytol, [alpha]-ionone, [beta]-ionone, ursolic acid, oleanolic acid and betulinic acid. The four last compounds were proven to be inhibitors of trypanosomal GAPDH, which may in part explain these antitrypanosomal activities.
[c] 2012 Elsevier GmbH. All rights reserved.
Human African trypanosomiasis or sleeping sickness is a vectorborne parasitic disease whose vector is Trypanosoma brucei, a protist parasite transmitted to humans by tsetse flies (Glossina genus). Sleeping sickness which may evolve into a neurological disease and be lethal, threatens millions of people in 36 countries in sub-Saharan Africa (World Health Organisation (WHO) 2012). The number of actual cases is currently estimated at 30,000 (Drugs for Neglected Diseases initiative 2011).
Resistance, toxicity and variable efficacy between strains or species of most of the drugs used as well as, for some of them, the need for a long course of parenteral administration show how the search for new antitrypanosomal compounds is needed, particularly from plants used in traditional medicine, as a source of new leads with new mechanisms of action (Hoet et al. 2004b).
In our search for more effective drugs against Trypanosoma brucei and as a continuation of our investigations on traditional plants used as antiparasitics in Benin (Bero et at. 2011), a special attention was devoted to Keetia leucantha which is a West African tree used in traditional medicine to treat parasitic diseases.
The dichloromethane extract of leaves of K. leucantlia was previously shown to have in vitro antitrypanosomal activity ([IC.sub.50] = 24.4 [micro]g/ml on Trypanosoma brucei brucei) with a good selectivity (SI = 2.7) (Bero et at. 2011). As triterpenic acids were identified in the twigs, we searched for this type of compounds in the leaves extract (Bero et al. 2011) but also analyzed its essential oil (obtained by hydrodistillation) as its components may also be found in dichloromethane extracts, and evaluate its antitrypanosomal activity.
One of the targets to fight trypanosomiasis is glycolysis (Verlinde et al. 2001), as the bloodstream form of Tbb is exclusively dependent on the breakdown of glucose by this process as its unique source of ATP production. Indeed, compounds which inhibit glycolysis have been shown to have antitrypanosomal activity (Bakker et al. 1999; Fairlamb et al. 1977). So we decided to evaluate seven constituents of the essential oil and the major triterpenic acids identified for their antitrypanosomal activity on T. b. brucei mammalian bloodstream forms and procyclic-insect forms with regard to a possible activity on trypanosome glycolysis. The compounds which, in this respect, appeared most promising were also assessed for an inhibitory activity on one of the glycolytic enzymes of T. brucei, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
Material and methods
Leaves of Keetia leucantha (K. Krause) Bridson (syn. Plectronia leucantha Krause) were collected and dried in Benin in November 2009. Voucher specimen was identified and deposited at the Herbarium of the National Botanic Garden of Belgium, at Meise bearing the number BRO000005087129.
Identification of terpenic acids in dichloromethane leaves extract
The identification of these compounds was realized with a LC-MS/MS system consisting of a Thermo Accela pump, autosam-pier, photodiode array detector and Thermo Scientific LTQ orbitrap XL mass detector. The column used was a Phenomenex Licrospher Cl 8 column, 4 mm x 250 mm packed with 5 p.m particles. The flow rate was 800 III/min using a binary solvent system: solvent A, HPLC grade water-acetonitrile (9:1) 0.1% formic acid and solvent B, acetonitrile with 0.1% formic acid (0-5 min: 100% A, 70-85 min: 0% A, 86-96 min: 100% A). High-resolution MS was performed on a Thermo Scientific LTQ orbitrap XL mass spectrometer with APCI source in the positive mode. The following inlet conditions were applied: capillary temperature 250 C, APCI vaporizer temperature 400 C, sheath gas flow 20.00 u.a., auxiliary gas flow 5.00 u.a., sweep gas flow 5.00 u.a. Data acquisition and processing were performed with Xcalibur software. Injected standard compounds were ursolic acid (Sigma), oleanolic acid (Sigma) and betulinic acid (Sigma).
50g of dried powdered leaves were hydrodistillal during 5 hours in a Clevenger-type apparatus (yield: 0.02% (w/w)). The obtained oil was diluted in tert-butyl methyl ether (107 mg/ml) for analysis by GC-MS.
The GC-FID analysis was performed on a Thermo Finnigan Focus GC with a FID detector using a capillary column, CP Sil 8 CB low bleed MS (30 m x 0.25 [micro]m; film thickness: 0.25 [micro]m, Varian). Samples were injected in splitless mode under the following conditions: injector temperature, 250 [degrees]C; oven temperature program, 50 [degrees]C to 100 [degrees]C at 10 [degrees]C/min and held at 100 [degrees]C for 10 min, 100 [degrees]C to 250 [degrees]C at 10 [degrees]C/min and held at 250 [degrees]C for 30 min; carrier gas, helium at 0.8 ml/min; injection volume, 1 [micro]l.
The GC-El MS analysis was performed on a TRACE GC 2000 series ThermoQuest instrument equipped with an autosampler AS2000 ThermoQuest and interfaced to a TRACE MS mass spectrometer operating in the electron-impact mode (70 eV). Separations were performed on the same column and under the same conditions as described before.
The NIST/EPA/NIH mass spectral library of the GC-MS system was used for the identification of constituents. The linear retention indices (LRI) were determined relative to the retention times of a series of C7-C25 fatty acid methyl esters (FAME).
The relative proportions of the constituents of the essential oil were calculated by electronic integration of the FID peak areas using the normalization method.
Parasites, cells and media
Dichloromethane extract, essential oil and isolated compounds were evaluated for their antitrypanosomal activity in vitro against T. b. brucei (strain Lister 427) bloodstream forms and procyclic forms. Tbb bloodstream forms (Tbb BSF) were cultured in vitro at 37 [degrees]C with 5% C[O.sub.2] in HMI9 medium containing 10% heat-inactivated fetal bovine serum. Tbb procyclic forms, which correspond to the lifecycle stage living in the tsetse midgut, were cultured in vitro at 28 [degrees]C with 5% C[O.sub.2] in a semi-defined medium (SDM-79) supplemented with 15% heat-inactivated fetal bovine serum (Tbb PF) (Brun and Lun 1994).
In vitro antitrypanosomal activity and parasite motility
The in vitro test was performed as described by Hoet et al. (2004a). Suramin (a commercial antitrypanosomal drug) was used as a positive control with an initial concentration of 1 [micro]g/ml. First stock solutions of crude extracts, essential oils and compounds were prepared in DMSO at 20 mg/ml. The solutions were further diluted in medium to give 0.2 mg/rill stock solutions. Extracts, essential oils and distinct compounds were tested in eight serial 3-fold dilutions (final concentration range: 100-0.05 [micro]g/m1) in 96-well microtiter plates. All tests were performed in duplicate.
The motility of parasites was observed with ursolic acid, betulinic acid and oleanolic acid at a concentration corresponding to five times the IC50 after 15, 30, 45, 60, 90, 120, 180, 240, 360 min.
Inhibition of GAPDH activity
The glycolytic enzyme, GAPDH, of T. brucei was prepared by overproducing it in Escherichia coli. The strain harboring an expression plasmid of trypanosomal GAPDH has been constructed by Hannaert et al. (1995). The GAPDH enzyme possesses a polyhistidine tag allowing its purification by affinity chromatography. The enzyme was purified according to published procedures (Hannaert et al. 1994, 1995) with some modifications: the affinity column used was a Talon resin (Clontech) and the elution of the proteins from the column was performed with an imiclazole gradient. All fractions were collected and concentrated using an Amicon YM-10 filter and stored at 4 [degrees]C. Inhibitory activities of all tested compounds on GAPDH were determined by spectrophotometry following published procedures (Claustre et al. 2002; Gelpi et al. 1993; Willson et al. 1994). Compounds were dissolved in DMSO with stock solutions at a final concentration of 2 mg/ml. Compounds were incubated with GAPDH during 2 min before adding substrate. The reaction was started by the addition of substrate mix (cofactors and auxiliary enzymes). The composition of the reaction mixture was: 0.1 M triethanolamine hydrochloride (TEA) pH 7.4, 5 mM MgS[O.sub.4], 1 mM EDTA, 0.42 mM NADH, 6 mM glycerate-3-phosphate, 1 mM ATP, 1 mM NaHC[O.sub.3], 1 mM dithiothreitol (DTT) and 22.5 units 3-phosphoglycerate kinase (PGK). The activity was measured by following the oxidation of NADH at 340 nm; six measurements of absorbance were realized each 30 s during 3 min. The slopes of the curves were recorded and inhibitory activities were determined by the percentage of inhibition at 20 [micro]g/ml of compounds compared to a negative control (DMSO). A possible effect of the inhibitors on the absorbance of NADH was checked and no effects on the enzyme activity were observed with 1% of DMSO used as negative control.
Results and discussion
The essential oil of leaves of K. leucantha showed an antitrypanosomal activity with IC50 of 20.911g/1-n1, on Tbb BSF (Table 2). An IC50 of 39.7 [+ or -] 7.2 [micro]g/m1 was obtained for the antiplasmalial activity. The analysis of the composition and results obtained from GC-FID and GC-MS allowed identifying 42 constituents, representing 89.57% of the oil (Table 1). The unidentified peaks were at low amounts or not well separated. This is the first time that the composition of leaves essential oil of K. leucantha is reported. The essential oil from the leaves of another Can thium species, C. horridum, allowed to identify 26 compounds from which two of the four main constituents were also found in the essential oil of leaves from K. leucantha: n-hexadecanoic acid (8.32%) and (Z,Z,Z)-9,12,15-octadecatrien-1-ol (9.61%) (Chen et al. 2007).
Table 1 Chemical composition of the essential oil of Keetia leucantha. Compounds Percentage LRI in oil (a) (b) Caryophyllene 0.46 1179.4 [alpha]-lonone 0.27 1197.6 Ceranyl acetone 0.21 1223.6 [beta]-lonone 0.23 1257.9 2-Naphtlialenemethanol,decahydro 0.16 1269.1 -[alpha], [alpha].4a-trimetliyl- 8-methylene-,2-R-(2[alpha].4a [alpha],8a[beta])] Lauiric anhydride 0.45 1349.0 (--)- Spathulenol 1.94 1362.8 Caryophyllene oxide 0.46 1367.6 Ledol 2.35 1381.4 Cubenol 0.14 1411.0 Tetracyclo|188.8.131.52(2.5).0(l,8)] 0.51 1423.6 tridecan-9-ol,4,4-di methyl- tau-Muurolol 0.38 1429.1 1-Naphthalenol, 1,2,3.4.4a.7. 0.14 1431.5 8.8a-octahydro-1,6-dimethyl-4- (1-methylerhyl)-, [1R-(1[alpha]. 4[beta],4a[beta].8a[beta)] [alpha]-Cadinol 1.31 1441.7 Trans-Z-[alpha]-Bisabolene 0.03 1472.4 epoxide 7R,8R-8-Hydioxy-4-isopropylidene- 0.36 1478.7 7-methylbicyclo[5,3,1]undec-1-ene Tetradecanol = myristyl 0.62 1490.6 6-lsopropenyl-4,8a-dimethyl- 0.27 1514.0 1,2,3,5.6,7,8,8a-octahydro- naphthalen-2-ol Tetradecanoic acid (C14) 5.19 1557.0 Phytone 4.3 1617.8 Acetylenedicarboxylic acid. 0.46 1633.6 di-(-)-mentliyl- 1,2-Benzenedicarboxylicacid. bis 0.95 1641.1 (2-methyIpropyl) ester Pentadecanoic acid 1.68 1647.7 9.12.15 Octadecatrien-1-ol 0.51 1671.0 (Z, Z, Z) = [C.sub.18][H.sub.12]0 5,9,13 Pentadecatrien-2-one-6,10, 0.71 1686.9 14 trimethyl(E, E)- Farnesylacetone Hexadecanoic acid, methyl ester 0.39 1697.2 Isophytol 1.09 1717.0 Dibutyl phthalate 1.75 1736.0 n-Hexadecanoic acid (C16) 43.46 1781.0 Phytol 5.68 1889.5 Oleic acid(C18:1) 9.51 1932.6 Octadecanoica cid 2.03 1948.9 3,7.11.15-Tetramethyl-2-hexadecen 0.02 1981.5 -1-ol Heneicosane 0.29 2063.3 4,8.12.16-Tetrametrvyllieptadecan 0.16 2122.9 -4-ol Hexatriacontane 0.3 2260.8 1,2-Benzenedicarboxylicacid. 0.04 2301.6 diisooctyl ester Tetracosane 0.05 2359.7 Octacosane 0.34 2459.3 Heptacosane 0.11 nd Squalene 0.16 nd nd Squalene 0.16 nd nd, not determined; retention time of compounds superior to the retention time of the last FAME. (a.) percentage obtained by the normalization method. (b.) LRI indices are indices of linear retention times relative to C7-C25 FAME calculated with a method set up in the laboratory (Denayer and Tilquin 1994: Herent et al. 2007). Table 2 In vitro antitrypanosomal activities of essential oil, dichloromethane extract and compounds on Tbb BSF and Tbb PF ([IC.sub.50] - [micro]g/m1([micro]M)). Tbb BSF Tbb PF Dichloromethane extract 19.9 [+ or -] 0.9 10.9 [+ or -] 3.3 Essential oil 20.9 [+ or -] 12.6 >100 Phytol 19.1 [+ or -]2.3 >100 (64.5) Geranylacetone 16.2 [+ or -] 12.5 >100 (77.6) Oleic acid 64.3 [+ or -] 0.5 >100 (>100) Caryophyllene 41.2+18.7 >100 (>100) [alpha]-lonone 13.1+5.9 81.4 [+ or -] 26.3 (68.2) (>100) [beta]-lonone 10.5 [+ or -] 5.8 55.8 [+ or -] 5.7 (54.6) (>100) n-Flexadecanoic acid >100 >100 Ursolic acid 2.5[+ or -]0.5 6.5 [+ or -] 0.1 (5.5) (14.2) Oleanolic acid 7.3+3.0 86.4 [+ or -] 19.2 (16.0) (>100) Betulinic acid 11.2[+ or -]6.9 40.9 + 7.7 (24.5) (89.6) Suramin 0.11 [+ or -]0.02 -- (84.8) (a) Tbb BSF, bloodstream forms of Trypanosoma brucei brucei; Tbb PF, procyclic forms of Trypunosorno brucei brucei: ncl, not determined. (a.) nM.
The dichloromethane extract of leaves of K. leucantha was further analyzed by LC-MS and allowed the identification of three triterpenic acids: betulinic (not present in the twigs extract), oleanolic and ursolic acids.
Seven commercially available constituents of the essential oil as well as the three triterpenic acids were evaluated for their antitry-panosomal activities on Tbb BSF. [IC.sub.50]s are given in Table 2. The best activities were observed for the three triterpenic acids and, from the essential oil, for geranylacetone, phytol, [alpha]-ionone and [beta]-ionone. The two main constituents of the essential oil, n-hexadecanoic acid and phytol, have [IC.sub.50]s > 100 [micro]g/ml and of 19.1 [micro]g/ml, respectively.
Since glycolysis is considered as an interesting target for antitrypanosomal molecules (Chen et al. 2007), we realized a preliminary study to analyze if glycolysis could be a target for these compounds. For this purpose, we analysed the antitrypanosomal activities on Tbb procyclic forms (Tbb PF) (Table 2). Compounds which were more active on bloodstream forms (BSF) than procyclic forms (PF) of Tbb may act on glycolysis. Indeed, bloodstream forms which have no Krebs cycle or mitochondrial respiratory chain coupled to ATP synthesis are exclusively dependent on glycolysis for their ATP formation. In contrast, procyclic forms were less dependent on glycolysis for their free energy because of their ability of oxidative phosphorylation and the possibilities to use amino acids, notably proline, as source of energy (both glucose and amino acids are present in the SDM-79 medium). Results given in Table 2 indicate that all tested active pure compounds ([IC.sub.50] < 20 [micro]g/m1), namely ursolic acid (UA), oleanolic acid (OA), betulinic acid (BA), geranyl acetone (GA), phytol (P), [alpha]-ionone ([alpha]-I) and [beta]-ionone ([beta]-I) may inhibit glycolysis because they have better [IC.sub.50] values on BSF than on PF.
Betulinic acid has already previously been shown to inhibit T. brucei GAPDH, a specific glycolytic enzyme which catalyses the phosphorylation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate with NAD+ and inorganic phosphate, with an IC50 value of 240 [micro]M and to do so as a competitive inhibitor of this enzyme (Nyasse et al. 2009). Indeed, GAPDH is considered a validated and attractive target for the design of trypanocidal drugs because its homologous mammalian enzyme is only 45-48% identical (Verlinde et al. 2001). Designed adenosine analogs are currently the most potent and selective inhibitors known for trypanosomal GAPDH, as for example N-6-(1-napthalenemethyl)-2'-deoxy-2'-(3-methoxybenzamido)-adenosine with an [IC.sub.50] of 2 [micro]M (Aronov etal. 1999). These inhibitors block pyruvate production from glucose and stunt growth of cultured Trypanosoma and Leishmania species.
Analysis of the activity of the selected compounds on T. brucei GAPDH showed that UA, OA, BA and [beta]-1 (Fig. 1) inhibit the activity of the enzyme from 30.4 to 72.6% at 20 [micro]g/m1 compared to the solvent (Table 3).
Table 3 Inhibition of trypanosomal GAPDI-1 activity (at 20 [micro]G/ml, in % compared to the solvent considered as 0%). Inhibition of GAPDH activity Phytol 6.8 [+ or -] 0.7 [alpha]-lonone 0.0 [+ or -] 7.2 [beta]-lonone 32.6 [+ or -] 3.4 Ursolic acid 30.4 [+ or -] 10.0 Oleanolic acid 72.6 [+ or -] 4.6 Betulinic acid 30.6 [+ or -] 5.6
The effect of some inhibitors was also tested on the motility of parasites, on the basis that the parasites are highly motile and completely dependent on glycolysis for their ATP production, and do not have any energy storage material. Therefore, any inhibition of glycolysis would immediately lead to reduction of the motility. The three triterpenic acids were tested at a concentration corresponding to 5 x [IC.sub.50]. OA, BA and UA stopped the movements of Tbb after respectively, 120, 240 and 360 min, providing another indication for a possible effect on glycolytic enzymes that leads to a decrease of ATP synthesis (Bisaggio etal. 2006).
A previous report showed that phytol had antiplasmodial activity with [IC.sub.50] values of 2.5 and 3.[micro]g/ml, respectively, on PoW and Dd2 strains of P. falciparum (Kohler et al. 2002). Phytol, geranyl acetone and [beta]-ionone were previously described for their antitry-panosomal activity with [IC.sub.50]s of 6.2, 5.2 and 5.514/ml on Tbb BSF (Hoet et al. 2006). In this study, we report a selective activity of -ionone on Tbb BSF with an inhibitory activity on trypanosomal GAPDH of 32.6% at 20 [micro]g/m1(104.2 [micro]M).
We also report here the more selective antitrypanosomal activities of three triterpenic acids on bloodstream forms than on procyclic forms. Our results are in accordance with those of the literature (Hoet et al. 2007; Taketa et al. 2004). Moreover, we showed that the three triterpenic acids stopped relatively rapidly all the movements of T. brucei at a concentration corresponding at 5 x [IC.sub.50]. This result is similar to that obtained in another study on Trypanosoma cruzi, showing that ursolic and oleanolic acids stopped all the movements of cultured epimastigote forms (i.e. the insect stage of T. cruzi) at 40 and 250 [micro]g/ml after 48 h of incubation (Abe et al. 2002). We also showed that they inhibit at 20 pg/ml or 43.8 [micro]M the activity of GAPDH, a glycolytic enzyme of T. brucei, with the best percentage of inhibitory activity for oleanolic acid (72.63%).
Pentacyclic triterpenoids are also known to possess anti-inflammatory and anti-HIV activities and induce apoptosis (Fukushima et al., 2011: Jager et al., 2009). Moreover, they have been shown to inhibit several enzymes, including human topoisomerases 1 and II. The precise mechanism of action has not been elucidated yet, but it would be of interest to study the effect on trypanosomal topoisomerases because the GAPDH inhibitory activity cannot completely explain the antitrypanosomal activity of the three acids and [beta]-ionone which probably behave as multi-target drugs. One of these targets could be the glycolysis and especially trypanosomal GAPDH.
This paper reports for the first time the composition and antitrypanosomal activity of the essential oil of K. leucantha. Several of its constituents and three triterpenic acids present in the dichloromethane leaves extract showed a higher antitry-panosomal activity on bloodstream forms of Tbb as compared to procyclic forms. It is the case for geranyl acetone, phytol, [alpha]-ionone, [beta]-ionone, ursolic acid, oleanolic acid and betulinic acid. The four last ones were proven to be inhibitors of trypanosomal GAPDH. However inhibition of this glycolytic enzyme may only partially explain the antitrypanosomal activities of these compounds.
One of possible next steps will be to verify a possible synergism between ursolic acid and suramin. Indeed, phytopharmaceuticals can be used in combination with synthetic drugs as suramin, to increase its effects (Wagner 2011).
The authors are grateful to Mr Agabani (botanist at the University of Abomey-Calavi, Cotonou, Benin), Dr Gbaguidi Fernand, Dr Gbenou Joachim, Dr Califon Habib and Prof. Mansourou Moudachi-rou (Centre Beninois de la Recherche Scientifique et Technique, Cotonou, Benin and Laboratoire de pharmacognosie et huiles essentelles UFR pharmacie, Faculte des Sciences de la Sante, Universite d'Abomey Calavi, Cotonou, Benin) for plant collections as well as Prof. Elmar Robbrecht and Olivier Lachenaud (botanists at the National Botanic Garden of Belgium, Meise, Belgium) for clarifying botanical information.
We wish to thank Marie-Christine Fayt and Ramazan Colak for skillful technical assistance. We would also like to thank Nathalie Chevalier from the TROP unit (de Duve Institute) for help to purify GAPDH.
The authors gratefully thank the Belgian National Fund for Scientific Research (FNRS) (FRFC 2.4555.08), the Special Fund for Research (FSR) and the faculty of medicine of UCL.
* Corresponding author. Tel.: +32 2 764 7292; fax: +32 2 764 7293. E-mail address: firstname.lastname@example.org (J. Bero).
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J. Beroa (a) *, C. Beaufaya (a), V. Hannaert (b), M.-F. Herent (a), P.A. Michels (b), J. Quetin-Leclercq (a)
(a) Universite catholique de Louvain, Louvain Drug Research Institute, Pharmacognosy Research Group, Avenue E. Mounier, B1.72.03, B-1200 Brussels, Belgium
(b) Universite catholique de Louvain. de Duve Institute, Research Unit for Tropical Diseases, Avenue Hippocrate 74, B1.74.01, B-1200 Brussels, Belgium
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|Author:||Bero, J.; Beaufay, C.; Hannaert, V.; Herent, M.-F.; Michels, P.A.; Quetin-Leclercq, J.|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Feb 15, 2013|
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