New findings on Simalikalactone D, an antimalarial compound from Quassia amara L. (Simaroubaceae)

Experimental Parasitology 130 (2012) 341–347

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New findings on Simalikalactone D, an antimalarial compound from Quassia amara L. (Simaroubaceae) Stéphane Bertani a,b,1, Emeline Houël c, Valérie Jullian d,e, Geneviève Bourdy d,e, Alexis Valentin d, Didier Stien c,f, Eric Deharo d,e,⇑ a

USM0307, Laboratoire de Parasitologie Comparée et Modèles Expérimentaux, Muséum National d’Histoire Naturelle (MNHN), Paris, France Department of Biochemistry, University of California Riverside (UCR), Riverside, CA, USA UMR ECOFOG, Centre National de Recherche Scientifique (CNRS), Cayenne, France d UMR152 PHARMA-Dev, Université Paul Sabatier (UPS), Toulouse, France e UMR152 PHARMA-Dev, Institut de Recherche pour le Développement (IRD), Toulouse, France f Institut de Chimie des Substances Naturelles (ICSN), Centre National de Recherche Scientifique (CNRS), Gif-sur-Yvette cedex, France b c

a r t i c l e

i n f o

Article history: Received 24 April 2010 Received in revised form 10 February 2012 Accepted 13 February 2012 Available online 21 February 2012 Keywords: Antimalarial Simalikalactone D Quassinoid Plasmodium Quassia amara

a b s t r a c t Quassia amara L. (Simaroubaceae) is a species widely used as tonic and is claimed to be an efficient antimalarial all over the Northern part of the Amazon basin. Quassinoid compound Simalikalactone D (SkD) has been shown to be one of the molecules responsible for the antiplasmodial activity of a watery preparation made out of juvenile fresh leaves of this plant. Because of its strong antimalarial activity, we decided to have a further insight of SkD pharmacological properties, alone or in association with classical antimalarials. At concentrations of up to 200 lM, we showed herein that SkD did not exert any apoptotic or necrotic activities in vitro on lymphoblastic cells. However, an antiproliferative effect was evident at concentrations higher than 45 nM. SkD was inefficient at inhibiting heme biomineralization and the new permeability pathways induced by the parasite in the host erythrocyte membrane. With respect to Plasmodium falciparum erythrocytic stages, SkD was almost inactive on earlier and later parasite stages, but potently active at the 30th h of parasite cycle when DNA replicates in mature trophozoites. In vitro combination studies with conventional antimalarial drugs showed that SkD synergizes with atovaquone (ATO). The activity of ATO on the Plasmodium mitochondrial membrane potential was enhanced by SkD, which on its own had a poor effect on this cellular parameter. Ó 2012 Elsevier Inc. All rights reserved.

1. Introduction French Guiana is located on the Northeast coast of South America, where the humid tropical climate brings perfect conditions for persistent transmission of malaria all year long (Esterre et al., 2009). Although access to the healthcare system is easy and free in this French overseas department, people still use herbal antimalarial remedies, either alone or in combination with allopathic drugs (Vigneron et al., 2005). One of the most popular remedies is made out of Quassia amara L. (Simaroubaceae) leaves, sometimes referred to ‘‘Cayenne’s quinquina’’ due to its bitter taste and its febrifuge properties. In a previous study, we isolated Simalikalactone D (SkD), a quassinoid responsible for the antimalarial properties of a Q. amara from juvenile fresh leaves (Bertani et al., 2006). Later, ⇑ Corresponding author. Address: UMR152 IRD–UPS, Faculté de Pharmacie, 35 chemin des maraîchers, 31062 Toulouse cedex 9, France. Fax: +33 5 62 25 98 02. E-mail addresses: [email protected], [email protected] (E. Deharo). 1 Current address: UMR152 IRD–UPS, Institut de Recherche pour le Développement (IRD), Universidad Peruana Cayetano Heredia (UPCH), Lima, Peru. 0014-4894/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2012.02.013

we isolated from this plant a new antimalarial quassinoid compound, Simalikalactone E (SkE), which was slightly less active than SkD on Plasmodium falciparum malaria parasite (Cachet et al., 2009). Quassinoids are highly oxygenated triterpenes known to inhibit the growth of cultivated P. falciparum at nanomolar concentrations and also known to be active in vivo against rodent malaria with doses ranging from 0.7 to 18 mg/kg/day (Phillipson et al., 1993; Kuo et al., 2004; Muhammad et al., 2004). Because their therapeutic indices were thought to be unfavorable, quassinoids were not seriously considered for use as antimalarial drugs. Nonetheless, the wide traditional consume of Q. amara prompted us to deepen the pharmacological potential of SkD. In this present study, we report the antimalarial activity and pharmacological properties of SkD, against P. falciparum and lymphoblastic cells in vitro, and murine Plasmodium yoelii in vivo in an effort to better define the mechanisms involved in the SkD antiparasitic and toxic activities. We studied therefore the SkD effects on the biomineralization of heme, on malaria-infected red blood cell membrane permeability, on P. falciparum mitochondria. The combinational impact of classical antimalarial drugs with SkD was also determined.

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2. Materials and methods 2.1. Materials SkD (MW: 478.5) was isolated from Q. amara as described previously, dissolved in dimethylsulfoxide (DMSO), and stored at 20 °C (Bertani et al., 2006). RPMI 1640 medium, as well as, penicillin, streptomycin, and gentamycin were obtained from Sigma– Aldrich (France). Human and fetal bovine serums were purchased from Marcopharma Laboratories Ltd. (Denmark) and Sigma–Aldrich (France), respectively. Antimalarial drugs were supplied by Sigma–Aldrich (France) for amodiaquine (AMO), chloroquine (CQ), doxycycline (DOX), and quinine (Q); GlaxoSmithKline (France) for atovaquone (ATO), cycloguanil (CYC), and halofantrine (HALO); Cambrex Corporation (France) for artemether (ART); Novartis (Switzerland) for lumefantrine (LUM); and Roche (Switzerland) for mefloquine (MEF). [3H]-hypoxanthine was obtained from Amersham Life Sciences (United Kingdom). Doxorubicin, 3,3-dihexyloxacarbocyanine iodide (DiOC6), propidium iodide (PI), and uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) were purchased from Sigma–Aldrich (France). Annexin V/ fluorescein isthiocyanate (FITC) from BD Pharmingen (France). LasilixÒ furosemide was obtained from Aventis (France). Other compounds were supplied by Sigma Chemical Co. (France). 2.2. In vitro parasite and cell cultivation The CQ-resistant P. falciparum FcB1 strain was maintained in human red blood cells as described by Trager and Jensen (1976) using RPMI 1640 medium supplemented with 10% heat-inactivated human serum, 10 mM glucose, and 25 mM sodium bicarbonate. Parasites were maintained in candle jars at 37 °C in humidified atmosphere. The Raji line of lymphoblast-like cells was maintained in 2 mM L-glutamine-enriched RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 lg/ml streptomycin at 37 °C in a 5% CO2 humidified atmosphere. 2.3. In vitro antimalarial activity assays In vitro antimalarial activity was measured using the [3H]-hypoxanthine incorporation assay (Desjardins et al., 1979; Le Bras and Deloron, 1983). The sensitivity of P. falciparum erythrocytic stage was determined using the [3H]-hypoxanthine incorporation assay modified as follows: parasite cultures were enriched in trophozoites by Plasmagel flotation according manufacturer’s instructions (Laboratoire Roger Bellon) and were plated with 1.44 lCi/ml of [3H]-hypoxanthine 24 h after synchronization, when rings were predominant (1% parasitaemia and 2.5% hematocrit). Through one P. falciparum erythrocytic cycle, the cultures were sequentially subjected to 4 h pulses of tested compound serially diluted in [3H]hypoxanthine culture medium. After being pulsed, each culture was washed and then returned to normal [3H]-hypoxanthine culture conditions for an overall experiment of 48 h (12 periods of 4 h). Assays were conducted in triplicate during the same day and were performed once more later on other cultures of FcB1 malaria strain, following the same procedure. 2.4. In vitro drug interaction assay Criss-cross serial dilutions of SkD and conventional antimalarial drugs were performed in 96-well plates on non-synchronous parasite cultures. Combination effect was then measured using the [3H]-hypoxanthine incorporation-based assay for 48 h as described above. The IC50s were calculated for each drug alone and for its

respective criss-cross dilutions. The sum of 50% fractional inhibiP tory concentrations ( FIC50) were calculated with the following formula:

IC50 of drug A in mixture IC50 of drug B in mixture þ IC50 of drug A alone IC50 of drug B alone Results were analyzed as described by De Jongh (1961) and Berenbaum (1977, 1978). Isobolograms were then constructed from the FIC50s of SkD and classical antimalarials. A straight line P represents additive effect ( FIC50 = 1), a convex line represents P synergic effect ( FIC50 < 1), and a concave curve represents antagP onic effect ( FIC50 > 1). Isobolograms were constructed using the graphic function of the ExcelÒ program. Assays were performed twice on different cultures of FcB1 malaria strain. 2.5. Ferriprotoporphyrin IX biomineralization inhibition test (FBIT) FBITs were performed as described previously (Deharo et al., 2002). Briefly normal non-sterile flat bottom 96-well plates were incubated at 37 °C for 18–24 h with a mixture containing: 50 ll of SkD serially diluted, 50 ll of 0.5 mg/ml of haemin chloride freshly dissolved in DMSO, and 100 ll of 0.5 M sodium acetate buffer pH 4.4 (final pH 5). Then the plates were centrifuged at 1600g for 5 min, supernatants were discarded and remaining pellets, consisting of precipitate of b-hematin, were washed twice with DMSO. Pellets were dissolved in 150 ll of 0.1 M NaOH for spectroscopic quantification at 405 nm on microplate ELISA reader. Assays were conducted in triplicate and DMSO solvent and CQ were used as the negative and positive controls, respectively. 2.6. Alterations in membrane permeability assay The effect of drugs on the permeability pathways induced by the malaria parasite was evaluated as described previously (Kutner et al., 1982) and modified as follows: Trophozoite-enriched cultures (parasitaemia >10% (Deharo et al., 1994)) were washed twice with saline buffer (HEPES 20 mM, NaCl 150 mM; pH 7.4, 304 mosM), and resuspended in the same buffer at 50% hematocrit. Afterward, 100 ll of cell suspension were transferred in EpendorffÒ tubes with 900 ll of an isotonic solution of sorbitol (300 mM, 20 mM; pH 7.4, 345 mosM) with increased concentrations of tested compound. The mixture was incubated at 37 °C for 15 min and spun at 3000g for 10 s and 150 ll supernatant aliquots were read in triplicates on micro-ELISA reader for hemoglobin content at 405 nm. LasilixÒ furosemide was used as the positive control. The same procedure was performed with healthy erythrocytes (external control). Assays were conducted in triplicate during the same day on the same culture and were performed once more later on another culture of the same malaria strain, following the same procedure. 2.7. Flow cytometric assays for mitochondrial membrane potential (DWm) Measuring the effect of various compounds either alone or in different combinations on the DWm was carried out using the method described in detail by Srivastava et al. (1997). Briefly, 5  106 trophozoite-infected red blood cells were incubated with 20 nM DiOC6 for 20 min at 37 °C (Deharo et al., 1994). Stained cells were aliquoted and incubated for 20 min with no compound (negative control), 0.5 lM ATO, 1 lM SkD, or 0.5 lM ATO and 1 lM SkD. Then, the parasitized cells were subjected to fluorescenceactivated cell sorter (FACS) analysis. The results were calculated from the mean fluorescence of 104 cells in histograms (FL1H; DiOC6 emission wavelength: 504 nm). In each experiment

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fluorescence intensity measurements were carried out in the presence or absence of probe to assess the baselines. Results were expressed as percent inhibition of fluorescence intensity, using the measurement in the presence of CCCP as a reference for 100% DWm collapse (Srivastava et al., 1997). Assays were conducted in triplicate during the same day and were performed once more later on other cultures of FcB1 malaria strain, following the same procedure.

membrane channels are deleterious for the parasite as it depends on those transport pathways to metabolize nutrients from plasma (Kutner et al., 1982). We did not see any impact on the membrane permeability of infected nor non-infected red blood cells with doses of SkD up to ten times the IC50 on Plasmodium (data not shown).

2.8. In vitro cytotoxicity and cytoproliferation studies

We studied then the impact of SkD on synchronized radiolabeled culture of P. falciparum to determine what was the most sensitive erythrocytic stage of the parasite. Arnot and Gull (1998) showed that during P. falciparum intra-erythrocytic cycle protein synthesis increases quickly while the DNA synthesis peaks between the 20th and the 38th h. Afterwards, DNA, RNA, and protein synthesis decrease dramatically with schizont stage announcing the end point of the cycle with the appearance of segmented, condensed merozoites (Fig. 1A). We thus subjected our culture to pulses of SkD every 4 h for 48 h. The IC50s obtained with parasites treated from the beginning of the experiment to the 22nd h and from the 26th h to the end point of the experiment were higher than 30 nM. The IC50 dropped to 10 nM at the 30th h when the production of plasmodial DNA is maximal and protein synthesis still elevated (Fig. 1B). Thus, SkD acts in a very peculiar moment of Plasmodium growth, being almost inactive on young and old cells but potently active on mature cells at the DNA replication stage (Fig. 1A and B). This result correlates with the stage activity of the SkE published previously (Cachet et al., 2009). Mata-Greenwood et al. (2001) showed that some quassinoids are able to inhibit DNA synthesis with greater efficacy when these molecules possess a C-15 ester side chain; that is the case for SkD (Fig. 2).

106 Raji cells plated in 24 well plates were exposed to different concentrations of SkD, and incubated for 24 h at 37 °C with 5% of CO2. In FACS, FITC was used to quantitatively determine the percentage of cells undergoing apoptosis and PI was used to distinguish viable from nonviable cells (Koopman et al., 1994). At least 104 gated events were analyzed using the CellquestÒ software to determine the frequencies of the different staining. Doxorubicin was used as control for cytotoxicity. Assays were performed in triplicate. In parallel, cells were stained with trypan blue (0.2% final concentration, 3 min) and cell proliferation was evaluated in counting, for each concentration of SkD tested, the living cells not dyed. The curve of cell concentrations in function of SkD concentrations was then plotted. 2.9. Statistical analysis

DWm data statistical analysis was performed using Kruskal– Wallis test (SASÒ 9.1 software) with a threshold P value set to 0.05 in order to test the null hypothesis.

3.3. Sensitive stage

3. Results and discussion

3.4. Combination of SkD with conventional antimalarials

3.1. Previous results

An additive effect was observed for AMO, ART, CQ, CYC, DOX, HALO, LUM, MEF, and Q (Fig. 3A, B, D–J). Interestingly, the combination of SkD with ATO (ATO–SkD) displayed a synergic effect (Fig. 3C). The ATO–SkD IC50 required only 1/4 the concentration of ATO and 1/3 the concentration of SkD necessary for their individual IC50 levels. ATO is thought to inhibit electron transport through the parasite mitochondrial cytochrome bc1 complex and to affect the mitochondrial membrane potential (Mather et al., 2005). Resistance occurs readily when ATO is used alone, thus it is commercialized as a fixed-ratio combination with the antifolate drug proguanil. Although the mechanism of such interaction has not been clearly elucidated, Srivastava and Vaidya (1999) have showed that proguanil, which itself has no effect on the mitochondrial respiratory chain, enhances the ability of ATO to collapse mitochondrial membrane potential, when used in combination. Brusatol (Fig. 2), a SkD-related structure molecule, increases the concentration of reduced mitochondrial electron-transport cofactors, thereby blocking aerobic respiration (Eigebaly et al., 1979). Cuendet and Pezzuto (2004) showed that bruceantin (Fig. 2) had apoptotic properties against HL-60 promyelotic by activation of caspase and mitochondrial pathways of apoptosis. Other quassinoids have also been shown to induce mitochondrial membrane depolarization (Rosati et al., 2004). Thus, we measured the impact of SkD, ATO, and ATO–SkD on the DWm using an in vivo DiOC6 staining of P. falciparum mitochondria. At the tested dose, ATO inhibited 13% of the fluorescence intensity (Fig. 4). This result is consistent with findings of other studies, demonstrating that ATO alone has a moderate impact on DWm of P. falciparum (Srivastava et al., 1997). In the same conditions, SkD alone had almost no impact on DWm, inhibiting only 5% of the fluorescence intensity (Fig. 4). Interestingly, SkD was able to significantly enhance the action of ATO on the parasite mitochondria when both drugs were

In a previous study, we showed that a remedy made from Q. amara leaves was active in vitro (IC50 = 8.9 lg/ml) against the CQresistant P. falciparum W2 strain (Bertani et al., 2005), a fact previously showed by Ajaiyeoba et al. (1999). Working further on this species, we isolated and characterized the quassinoid compound SkD (Bertani et al., 2006, 2007). In our experience, on the CQ-resistant P. falciparum FcB1 strain, SkD was 12 times more active than CQ (Table 1). These data were consistent with previous reported values (O’Neill et al., 1986; Cabral et al., 1993; Muhammad et al., 2004). Our study was then completed with the isolation of SkE, which was less active in vitro than SkD (Cachet et al., 2009). 3.2. Heme biomineralization and permeability pathways CQ and 4-aminoquinolines have been shown to directly interact with heme biomineralization (Deharo et al., 2002), a crucial metabolic event, taking place in Plasmodium during its erythrocytic stage. We thus measured the impact of SkD on this process, but no effect was evident (data not shown). Plasmodium is known to induce new permeability pathways in membrane of malaria-infected erythrocyte. Alterations of these

Table 1 Antimalarial and cytotoxic activities of SkD and CQ on P. falciparum, P. yoelii nigeriensis, and Raji lymphoblast-like cell line.

SkD CQ

P. falciparum FcB1 IC50 (nM)

P. yoelii nigeriensis ED50 (mg/kg)

Raji cells IC50 (nM)

10 ± 2 120 ± 10

0.7 ± 0.1 2.3 ± 0.3

>500 >500

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Fig. 1. Evolution of the antimalarial activity of SkD during the course of the red blood cell cycle of P. falciparum. (A) Represents in parallel the erythrocytic morphological development of Plasmodium from ring stage to schizont, and the evolution of RNA, protein, and DNA synthesis ratio (adapted from Arnot and Gull, 1998). (B) Corresponds to the follow-up of the SkD IC50 on FcB1 malaria strain, monitored every 4 h during 48 h. The slim gray strip highlights the IC50 of SkD obtained in asynchronous culture conditions on FcB1 strain.

used in combination (Fig. 4) (P < 0.0001). The fluorescence intensity was then attenuated of more than 40% demonstrating synergic effect on the parasite mitochondria of the ATO–SkD combination (Fig. 4). This potentiation effect of ATO–SkD on the DWm correlates well with the synergic antimalarial effect observed when ATO and SkD are used together, as described above (Figs. 3C and 4). The action of ATO–SkD on the parasite mitochondria may explain the strong antimalarial activity of this combination. 3.5. Combination of SkD with Q. amara contents

Fig. 2. Chemical structures of SkD, brusatol, and bruceantin.

When given alone, 3.7 mg/kg/day of SkD are necessary to inhibit 50% of the parasite growth in vivo on murine malaria, i.e. a dose very similar to that of CQ (ED50 = 2.6 mg/kg/day) (Bertani et al., 2006). This dose is almost ten times more elevated than the SkD concentration found in the infusion displaying the same activity (Bertani et al., 2007; Houël et al., 2009). Thus, something probably enhances the antimalarial activity of SkD in the watery preparation. This phenomenon has also been described with the Chinese

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P P Fig. 3. Isobolograms of the interactions of SkD with classical antimalarials on P. falciparum FcB1 strain (Abscissas: allopathic drug FIC50; Ordinates: SkD FIC50). The diagonal line represents additive effect, while convex and concave plotted curves represent synergism and antagonism effects, respectively.

McIntosh and Olliaro, 2001). Some authors have suggested the role of synergetic substances to explain this phenomenon (Elford et al., 1987; Chen Liu et al., 1992). For that reason, we studied on Plasmodium in culture the interactions of quassin and picrasin (two quassinoids present in the traditional preparation (Houël et al., 2009)) with SkD. The results indicated that quassin alone was inactive while picrasin alone has an IC50 of 0.3 lg/ml, i.e. 60 times less active than SkD, but none of these products potentialized the antimalarial activity of SkD (data not shown). Therefore, further research should be then conducted to identify the reasons why the infusion is proportionally more active in vivo than SkD alone (also discussed in Deharo and Ginsburg, 2011). 3.6. Cytotoxicity

Fig. 4. Activity of ATO (0.5 lM), SkD (1 lM), and ATO–SkD (0.5/1 lM ratio) on the mitochondrial P. falciparum DWm. Inhibitions of fluorescence intensity were statistically different for synergic ATO–SkD and both ATO and SkD used alone (P < 0.0001; N = 6). Error bars represent the standard error of the mean.

traditional preparation of Artemisia annua L. (Asteraceae), which contains less than 20% of the usually recommended daily dose of artemisinin in conventional treatments (Räth et al., 2004;

In a phase I clinical study conducted in 66 patients with various types of advanced solid tumors, Bedikian et al. (1979) administered bruceantin up to 4.5 mg/m2/day for 5 days (i.e. 0.15 mg/kg). Patients reported that hypotension, nausea, and vomiting were common side effects at higher doses, but hematological toxicity was moderate to insignificant and manifested itself as thrombocytopenia. Moreover, it has been shown that the sensitivity to bruceantin varies between cell lines and the same thing occurred with SkD: 6.3 nM on epidermoid carcinoma KB cells, 2 lM on cervical carcinoma HeLa cells, 10 lM on primate kidney epithelial Vero cells, 0.5 lM on melanoma SK-MEL cells, breast carcinoma BT-549 cells,

Fig. 5. FACS analysis of the apoptotic and cytotoxic effects of SkD and doxorubicin. FITC (FL1-H; abscissas) and PI (FL2-H; ordinates) labeling are presented for Raji cells alone (negative control) (A), treated with 10 lM doxorubicin (positive control) (B), and treated with 200 lM SkD (C). Living cells [FITC()/IP()] are distributed in lower left panel (LF); apoptotic cells [FITC(+)/IP()] are distributed in lower right panel (LR); and necrotic cells are distributed [IP(+)] in upper panel (U). (A) LF: 92%; LR: 5.6%; U: 2.4%; (B) LF: 1%; LR: 0.4%; U: 98.6%; (C) LF: 82%; LR: 10.5%; U: 7.5%.

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and ovarian carcinoma SK-OV cells (O’Neill et al., 1986; Cuendet and Pezzuto, 2004; Muhammad et al., 2004; Fukamiya et al., 2005). In our experience and for the first time, FACS analysis revealed that the percentages of the Raji B cells undergoing apoptosis and necrosis exposed to 200 lM, i.e. 20,000-fold higher than the malarial IC50, were not significantly different than the ones recorded for untreated control cells (Fig. 5A–C). However, we observed an antimitotic activity on Raji cells for concentrations of SkD higher than 45 nM. The wide range of activity on different cellular lines is reflected in the plethora of biological activities displayed by the quassinoids, including anti-tumor, anti-viral, and anti-inflammatory (Hall et al., 1983; Apers et al., 2002; Murakami et al., 2004; Almeida et al., 2007). Many mechanisms have been evoked: apoptosis induction, inhibition of de novo synthesis of purine bases, inhibition of DNA-RNA synthesis, and inhibition of the aerobic respiration (Eigebaly et al., 1979; Hall et al., 1979; Kirby et al., 1989; Rosati et al., 2004). Nevertheless, the most accepted mechanism remains the inhibition of protein synthesis (Liao et al., 1976; Kirby et al. 1989; Liou et al. 1982; Fukamiya et al., 2005). Some authors have speculated that quassinoids inhibit protein synthesis via interference at the peptidyltransferase site, preventing the first round of peptide bond formation prior to polysome formation, this inhibition being reversible (Fresno et al., 1978; Willingham et al., 1981; Willingham et al., 1984). 4. Conclusions From Northeast of the Amazon up to Central America, Q. amara is the most common herbal traditional remedy for treatment against malaria and could be considered as the South American A. annua. Although Q. amara is used daily by thousands of people all over Amazonia, the toxicity attributed to quassinoids impairs any commercial development of antimalarial treatment based on the use of those molecules. Nevertheless, the wide utilization of quassinoid containing plants in conjunction with experimental evidence should change our perception of the quassinoid family. In the case of SkD, it may be considered as a potential adjuvant for ATO reducing both its inherent toxicity and the ATO toxicity itself and could also delay the emergence of resistance to ATO in a drug combination approach. Further studies on the in vivo pharmacology of SkD are required to confirm its interest in malaria therapy. In parallel, Q. amara opens the doorway in the search of ‘‘molecules beside the molecules’’ (Deharo and Ginsburg, 2011), compound(s) without direct antimalarial property but able to enhance ten times the antimalarial activity of SkD, when prepared in the form of an infusion. Acknowledgments The authors thank Vincent Lacoste (Institut Pasteur de la Guyane) for providing Raji cell lines; David Parker Jr. and Brian Gadd (University of California, Riverside) for the critical reading of the manuscript. The authors have written this article under the auspices of CaP (Chemotherapy against Parasites/Consortium antiParasitaire). References Ajaiyeoba, E.O., Abalogu, U.I., Krebs, H.C., Oduola, A.M.J., 1999. In vivo antimalarial activities of Quassia amara and Quassia undulata plant extracts in mice. Journal of Ethnopharmacology 67, 321–325. Almeida, M.M.B., Arriaga, A.M.C., Dos Santos, A.K.L., Lemos, T.L.G., Braz-Filho, R., Curcino Vieira, I.J., 2007. Occurrence and biological activity of quassinoids in the last decade. Quimica Nova 30, 935–951. Apers, S., Cimanga, K., Vanden Berghe, D., van Meenen, E., Otshudi Longanga, A., Foriers, A., Vlietinck, A., Pieters, L., 2002. Antiviral activity of Simalikalactone D, a quassinoid from Quassia Africana. Planta Medica 68, 20–24.

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