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Leishmanicidal compounds and potent PPARγ activators from Renealmia thyrsoidea (Ruiz & Pav.) Poepp. & Endl. Billy Joel Cabanillas a,b,c, Anne-Cécile Le Lamer a,b,n, David Olagnier a,b, Denis Castillo d, Jorge Arevalo d, Céline Valadeau a,b,c, Agnès Coste a,b, Bernard Pipy a,b, Geneviève Bourdy a,b,c, Michel Sauvain a,b,c, Nicolas Fabre a,b a

Université de Toulouse III, UPS, PHARMA-DEV, UMR 152, 118 Route de Narbonne, F-31062 Toulouse Cedex 9, France IRD, UMR 152, F-31062 Toulouse Cedex 9, France c IRD, UMR 152, Mission IRD Casilla 18-1209, Lima, Peru d Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia, Avenida Honorio Delgado 430, San Martin de Porres, Lima, Peru b

art ic l e i nf o

a b s t r a c t

Article history: Received 29 April 2014 Received in revised form 4 September 2014 Accepted 6 September 2014

Ethnopharmacological relevance: Leaves and rhizomes of Renealmia thyrsoidea (Ruiz & Pav.) Poepp. & Endl. traditionally used in the Yanesha pharmacopoeia to treat skin infections such as leishmaniasis ulcers, or to reduce fever were chemically investigated to identify leishmanicidal compounds, as well as PPARγ activators. Methods: Compounds were isolated through a bioassay-guided fractionation and their structures were determined via detailed spectral analysis. The viability of Leishmania amazonensis axenic amastigotes was assessed by the reduction of tetrazolium salt (MTT), the cytotoxicity on macrophage was evaluated using trypan blue dye exclusion method, while the percentage of infected macrophages was determined microscopically in the intracellular macrophage-infected assay. The CD36, mannose receptor (MR) and dectin-1 mRNA expression on human monocytes-derived macrophages was evaluated by quantitative real-time PCR. Results: Six sesquiterpenes (1–6), one dihydrobenzofuranone (7) and four flavonoids (8–11) were isolated from the leaves. Alongside, two flavonoids (12–13) and five diarylheptanoids (14–18) were identified in the rhizomes. Leishmanicidal activity against Leishmania amazonensis axenic amastigotes was evaluated for all compounds. Compounds 6, 7, and 11, isolated from the leaves, showed to be the most active derivatives. Diarylheptanoids 14–18 were also screened for their ability to activate PPARγ nuclear receptor in macrophages. Compounds 17 and 18 bearing a Michael acceptor moiety strongly increased the expression of PPARγ target genes such as CD36, Dectin-1 and mannose receptor (MR), thus revealing interesting immunomodulatory properties. Conclusions: Phytochemical investigation of Renealmia thyrsoidea has led to the isolation of leishmanicidal compounds from the leaves and potent PPARγ activators from the rhizomes. These results are in agreement with the traditional uses of the different parts of Renealmia thyrsoidea. & 2014 Elsevier Ireland Ltd. All rights reserved.

Keywords: Renealmia thyrsoidea Zingiberaceae Leishmanicidal activity PPARγ Medicinal plants Peru

1. Introduction In South America, there are an estimated 75 species of Renealmia (Zingiberaceae), most of which are highly valued as medicinal plants, used for various purposes (Willis, 1985; Maas, 1997). n Corresponding author at: Université de Toulouse III, UPS, PHARMA-DEV, UMR 152, 118, Route de Narbonne, F-31062 Toulouse Cedex 9, France. Tel.: þ 33 5 62 25 68 48; fax: þ 33 5 61 55 43 30. E-mail address: [email protected] (A.-C. Le Lamer).

Aerial parts of Renealmia alpinia (Rottb.) Maas, Renealmia floribunda K. Schum., Renealmia guianensis Maas, Renealmia monosperma Miq. and Renealmia thyrsoidea are prepared in the form of a decoction, or are rubbed locally to help the healing of wounds. They can also be used in case of various dermatitis, such as scabies or other related fungal infections, as well as leishmaniasis ulcers (DeFilipps et al., 2004; Grenand et al., 2004). The same uses have also been reported for Renealmia alpinia rhizomes (Fenner et al., 2006). Ringworm is more specifically treated with Renealmia alpinia and Renealmia floribunda whole plants finally crushed in a paste (Ficker et al., 2003; DeFilipps et al., 2004). Whole plants or

http://dx.doi.org/10.1016/j.jep.2014.09.010 0378-8741/& 2014 Elsevier Ireland Ltd. All rights reserved.

Please cite this article as: Cabanillas, B.J., et al., Leishmanicidal compounds and potent PPARγ activators from Renealmia thyrsoidea (Ruiz & Pav.) Poepp. & Endl.. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.010i

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aerial parts of Renealmia alpinia, Renealmia monosperma, Renealmia guianensis, Renealmia floribunda and Renealmia thyrsoidea are prepared in the form of a bath, or rubbed all over the body to reduce fever (Schultes and Raffauf, 1990; DeFilipps et al., 2004; Grenand, et al., 2004), or when the person feels tired and wishes to recover a feeling of well-being (Grenand et al., 2004; Valadeau et al., 2009). Rhizomes of Renealmia guianensis, Renealmia floribunda, Renealmia monosperma or young shoots of Renealmia orinocensis Rusby, scratched and prepared in the form of a concentrated tea are said to treat malaria. Likewise, typhoid fever can be alleviated with a tea made from Renealmia alpinia leaves (DeFilipps et al., 2004). Finally, there is a strong convergence for the use of these Renealmia species (Renealmia alpinia, Renealmia asplundii Maas, Renealmia aromatica (Aubl.) Griseb., Renealmia cernua (Sw. ex Roem. & Schult.) J.F.Macbr., Renealmia nicolaioides Loes., and Renealmia thyrsoidea) as anti-ophidians (Bothrops or Crotalus species), as claimed in the northwest part of the Amazon. For use as anti-ophidians, which has been experimentally validated (Patiño et al., 2013), the whole plant is crushed, applied in form of a poultice and mixed with water to be consumed as tea (Schultes and Raffauf 1990; Brack Egg, 1999; Otero et al., 2000; DeFilipps et al., 2004). In the course of our ongoing research for natural antileishmanial compounds from Amazonian medicinal plants (Castillo, et al., 2007; Cabanillas et al., 2010; Cabanillas et al., 2012), two Renealmia species (Renealmia alpinia and Renealmia thyrsoidea) were collected on the bases of the Yanesha pharmacopoeia, an Amerindian Peruvian community (Bourdy et al., 2008). The rhizomes of both species displayed an interesting in vitro activity against Leishmania amazonensis axenic amastigotes (IC50 r10 μg/ml, Valadeau et al., 2009), and the leaves of Renealmia thyrsoidea exhibited a moderate activity (IC50 ¼19 μg/ml, unpublished results). Recently, our laboratory showed that macrophage response against Leishmania infection is characterized by the expression of specific surface receptors involved in the recognition and internalization of Leishmania parasites. Among them, mannose receptor (MR) and Dectin-1, which are up-regulated through the activation of the nuclear receptor PPARγ (Coste et al., 2003; Galès et al., 2010) favored the host response (Lefèvre et al., 2013). Interestingly, the genus Renealmia (Zingiberaceae) belongs to the same family as Curcuma longa, a well-known species for its diarylheptanoid derivatives such as curcumin, which is a potent activator of PPARγ (Xu et al., 2003; Jacob et al., 2007; Dong et al., 2011; Lin et al., 2012; Li et al., 2013). Altogether, these data led us to first investigate the antileishmanial properties of compounds isolated from the rhizomes and the leaves of Renealmia thyrsoidea, since this Renealmia species has not been chemically studied in the past and its antileishmanial potential remains uncovered. Then, we decided to focus our attention on diarylheptanoids from Renealmia thyrsoidea in order to explore whether these compounds are able to enhance PPARγ and key surface receptors expression. Results are discussed in relation with Renealmia thyrsoidea traditional medicinal uses.

2. Materials and methods 2.1. General experimental procedures The NMR experiments were recorded on a Bruker Avance 300 or 500 MHz instrument using DMSO-d6 or CDCl3. APCIMS and ESIMS spectra were obtained with a Thermo-Finnigans LCQ Deca XP Max ion trap mass spectrometer. CIMS spectra were performed using a Waters QToF Premier instrument. The analytical HPLC system consisted of a Merck Hitachi LaChrom L-7100 pump, a Merck Hitachi L7455 photodiode array detector, and a Pursuits

XRs-C18 column (250  4.6 mm2, 5 μm; Varian). Semi-preparative HPLC analysis was performed on a Microsorb 100-5 C18 Dynamax (250  10 mm2, 10 μm; Varian). Medium pressure liquid chromatography (MPLC) was carried out with a Büchi C-605 pump manager using SDS silica gel 60A (6–35 μm). For column chromatography, stationary phases of silica gel (Merck, 60A, 40–63 μm), C18 reversed-phase silica gel (Merck, LiChropreps, 60A, 40– 63 μm) and Sephadexs LH20 (Sigma Aldrich) were used. TLC were performed over silica gel 60 F254 precoated aluminum sheets (0.2 mm layer thickness, Merck). Spots were detected by spraying with 2% solution of vanillin in concentrated sulfuric acid and then heating. 2.2. Plant material The leaves and rhizomes of Renealmia thyrsoidea (Zingiberaceae) were collected in July 2007 from Loreto department in Peru and identified by Rodolfo Vázquez (Oxapampa Herbarium [HOXA], Missouri Botanical Garden, Oxapampa, Peru). Voucher specimens (CV622) are deposited at the Herbarium of Natural History Museum of Mayor de San Marcos University in Lima and at the HOXA in Oxapampa, Peru. 2.3. Extraction and isolation The dried powdered leaves of Renealmia thyrsoidea (840 g) were extracted three times with 90% EtOH (3  7 L) at room temperature and filtered. Evaporation of solvent under vacuum provided 48 g of crude extract. This extract was suspended in H2O/ MeOH (9:1, 1 L) and partitioned with cyclohexane (3  1 L) and ethyl acetate (3  1 L). Solvent was removed to afford cyclohexane (5.4 g), ethyl acetate (4.8 g), and aqueous (5.9 g) extracts. The ethyl acetate extract was chromatographed on silica gel by MPLC with a gradient of CH2Cl2–MeOH (v/v 1:0 to 0:1) to give eight fractions (Et1–8). Fractions Et1–Et4 showed the best activity when they were evaluated against axenic amastigotes of Leishmania amazonensis. Fraction Et1 was purified by column chromatography on RP-18 using a gradient of CH3CN:H2O (v/v 1: 9 to 6:4) to afford 8 (33 mg) and 10 (14 mg). Fraction Et2 was also purified using RP-18 and eluted with a solvent system of CH3CN:H2O (v/v 1:9 to 5:5) to generate five subfractions (Et2A1–5). Subfractions Et2A2 and Et2A4 were additionally chromatographed on silica gel eluted with C6H12:AcOEt (v/v 9:1 to 4:6) to give 9 (6 mg) and 7 (9 mg), respectively. Fraction Et3 was subjected to column chromatography on silica gel eluted with CH2Cl2:AcOEt (v/v 1:0 to 2:8) to generate six subfractions (Et3A1–6). Subfraction Et3A2 was further chromatographed on silica gel using a gradient of C6H12:AcOEt to afford four subfractions (Et3A2B1–4). Subfractions Et3A2B2 and Et3A2B3 were finally purified on Sephadex LH-20 using CH2Cl2 as eluent to yield 2 (15 mg) and 1 (18 mg), respectively. Subfractions Et3A3 and Et3A4 were fractionated on RP-18 using a gradient of MeOH:H2O (v/v 1:9 to 4:6) to give five (Et3A3B1–5) and six (Et3A4B1–6) subfractions, respectively. Purification by column chromatography on Sephadex LH-20 of subfractions Et3A3B2, Et3A3B4, Et3A4B2 and Et3A4B5 afforded compounds 3 (8 mg), 11 (3 mg), 6 (4 mg) and 5 (18 mg), respectively. Fraction Et4 was loaded in a RP18 column and eluted with MeOH:H2O (v/v 1:9 to 4:6) to generate five fractions (Et4A1–5). A final purification of Et4A2 on Sephadex LH-20 using a gradient of CH2Cl2:AcOEt (v/v 1:0 to 3:7) gave 4 (10 mg). The dried powdered rhizomes of Renealmia thyrsoidea (431 g) were extracted three times with 90% EtOH (3  4 L) at room temperature and then filtered. The solvent was concentrated to afford a crude extract (13 g). This extract was suspended in 10% MeOH (1 L) and then partitioned successively with cyclohexane (3  1 L), dichloromethane (3  1 L) and ethyl acetate (3  1 L).

Please cite this article as: Cabanillas, B.J., et al., Leishmanicidal compounds and potent PPARγ activators from Renealmia thyrsoidea (Ruiz & Pav.) Poepp. & Endl.. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.010i

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The ethyl acetate extract was concentrated under reduced pressure to yield a brown residue (1.95 g). This residue was subjected to column chromatography over Sephadex LH-20 and eluted with MeOH:H2O (v/v 5:5 to 8:2) to give six fractions (Et1–Et6). Fraction Et2 (573 mg) was chromatographed over RP-18 using a gradient of CH3CN:H2O (v/v 1:9 to 5:5) to generate seven fractions (Et2A1–7). Fraction Et2A3 (207 mg) was separated in two subfractions (Et2A3B1–2) by elution with CH3CN:H2O (3:7) and then further purified by semi-preparative HPLC using as mobile phase CH3CN: H2O (20:80 to 25:75) to afford 15 (63 mg, tR ¼7.2 min) and CH3CN: H2O (30:70 to 35:65) to afford 14 (29 mg, tR ¼ 6.5 min) and 16 (5 mg, tR ¼ 7.7 min). The dichloromethane extract was concentrated to dryness under reduced pressure and the residue (1670 mg) was subjected to a silica gel column using a CH2Cl2: MeOH gradient system (v/v 1:0 to 9:10) to give seven fractions (Dm1–7). Fraction Dm2 was separated in four fractions (Dm2A1–4) by elution with C6H12:AcOEt (v/v 9:1 to 7:3) over a silica gel column. Further purification of fraction Dm2A2 over the RP-18 column eluted with MeOH:H2O (6:4) gave 13 (15 mg). Fraction Dm3 (380 mg) was separated into three fractions (Dm3A1–3) by a silica gel column using a mixture of C6H12:AcOEt (5:5). Fraction Dm3A2 (160 mg) was passed through a RP-18 column eluting with MeOH:H2O (v/v 2:8 to 9:1) to give three fractions (Dm3A2B1–3). Purification by semi-preparative HPLC of fraction Dm3A2B1 (71 mg) with a gradient of CH3CN:H2O resulted in the isolation of 12 (11 mg) and 23 mg of a mixture that was further purified over a Sephadex LH-20 column eluted with MeOH:H2O (v/v 5:5 to 6:4) to yield 17 (13 mg) and 18 (9 mg). 2.4. Biological activities 2.4.1. Antileishmanial activity assay Antileishmanial activity was determined using the MTT micromethod (Estevez et al., 2007). The amastigotes of Leishmania amazonensis (strain MHOM/BR/76/LTB‑012) were grown in MAA/ 20 medium at 3271 1C, 5% CO2. The tested compounds dissolved in DMSO were added to the amastigotes seeded in 96-well microplates at a density of 2  105 cells/well. After 72 h of incubation, 10 mL of MTT (10 mg/mL) were added to each well and incubated for 4 h. The reaction was then stopped by addition of 100 ml of 50% isopropanol–10% sodium dodecyl sulfate. The plates were incubated under agitation for another 30 min and then read at 570 nm. Amphotericine B was used as reference compound. All experiments were repeated three times. 2.4.2. Cytotoxicity assay Macrophages were collected from the peritoneal cavities of six weeks old BALB/c male mice and placed in M199 medium enriched with 10% fetal calf serum (FCS). The cells were seeded in 96-well microplates and incubated for 24 h (37 1C, 5% CO2). Concentration in the wells was 7  104 cells/well approximately. Dilutions of tested compounds at the concentrations of 10, 1, and 0.1 μM in complete medium were then added to achieve a final volume of 100 μL. After 48 h of incubation (37 1C, 5% CO2) the number of viable cells was microscopically scored using 0.4% trypan blue solution in PBS (Castillo et al., 2007). 2.4.3. Intracellular infected macrophage assay Microplates containing the macrophages were prepared in medium M199 as described above. Then, the medium was replaced by the suspension of amastigotes using an infection ratio of 7/1 amastigotes/macrophages (Castillo et al., 2007) and incubated for 12 h. Solutions of the compounds to be tested were added to the cultures at various concentrations and maintained at 37 1C under 5% CO2 for another 48 h. Plates were fixed with

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methanol and treated with 10% Giemsa stain. The percentage of infected macrophages was determined microscopically. 2.4.4. Human monocytes isolation, differentiation into macrophages and treatments Human peripheral blood mononuclear cells were isolated from the blood of healthy volunteers by a density gradient centrifugation method on Lymphoprep (Abcys). Monocytes were isolated by adherence to plastic for 2 h in M-SFM at 37 1C, 5% CO2. Monocytes were cultured for 5–7 supplementary days in M-SFM containing 50 ng/mL of M-CSF (eBiosciences) to allow for differentiation into human monocytes-derived macrophages. Human monocytes-derived macrophages were incubated with rosiglitazone (5 mM) (Cayman Chemical) or with the compounds (5 mM) for 18 h in M-SFM at 37 1C, 5% CO2. 2.4.5. CD36, mannose receptor and dectin-1 mRNA expression by quantitative real-time PCR RNA and cDNA were prepared as previously described. Quantitative RT-PCR was performed on a LightCyclers 480 system using LightCyclers 480 SYBR GREEN I MASTER (Roche Diagnostics). β-actin was used as the invariant control. The sequences of primers are listed in Table 1. The N-fold differential expression of mRNA genes samples was expressed as 2ΔΔCt (Lefèvre et al., 2010).

3. Results 3.1. Chemical analyses The bioguided fractionation of the leaf ethanolic extract resulted in the isolation of (  )-oplopanone (1) (Takeda et al., 1966; San Feliciano et al., 1995), 1β,6α-dihydroxyeudesm-4(15)ene (2) (Hu et al., 1996), oplodiol (3) (Minato and Ishikawa, 1967; Herz and Watanabe, 1983), (  )-1β,4β,7α-trihydroxyeudesmane (4) (Sung et al., 1992), (5E)-germacra-5,10(14)-dien-1β,4β-diol (5) (San Feliciano et al., 1995), (þ)-4β,10α-aromadendranediol (6) (Anjaneyulu et al., 1995), 5,6-desmethylenedioxy-5-methoxy-aglalactone (7) (Salim et al., 2007), (2R,3R)-40 ,7-di-O-methyldihydrokaempferol (8), (2R,3R)-40 ,5,7-trimethoxydihydro-flavonol (9) (Lima and Polonsky, 1973; Islam and Tahara, 2000), 3,5-dihy- Q3 droxy-7,40 -dimethoxyflavone (10) (Erdtman et al., 1966), and 3,5,7,40 -tetramethoxyflavone (11) (Joseph-Nathan et al., 1981). In parallel, two flavanones (12, 13) and five curcuminoids (14–18) were isolated from the ethanol extract of the rhizomes of Renealmia thyrsoidea. These compounds were identified as naringenin (12), sakuranetin (13) (Grande et al., 1985), 3,5-dihydroxy-1,7-bis(4-hydroxyphenyl)-heptane (14), 3,5-dihydroxy-1(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-heptane (15) (Yokosuka et al., 2002), 1,7-bis(4-hydroxyphenyl)-5-hydroxy-3-heptanone (16) (Nomura et al., 1981; Ohta et al., 1985), 1,7-bis(4-hydroxyphenyl)-4E-hepten-3-one (17) (Nomura et al., 1981), and 1,7-bis (4-hydroxyphenyl)hepta-4E,6E-dien-3-one (18) (Ali et al., 2001). Table 1 Human primers sequences used in quantitative RT-PCR experiments. Human genes

Sequences

β-actin

Sense 50 CCT CAC CCT GAA GTA CC CA 30 Antisense 50 TGC CAG ATT TTC TCC ATG TCG 30 Sense 50 TGT AAC CCA GGA CGC TGA GG 30 Antisense 50 GAA GGT TCG AAG ATG GCA CC 30 Sense 50 TGA ACG GAA TGA TTG TGT AGC TT 30 Antisense 50 CAC GTT GGA AGA CGG TTT AGA AG 30 Sense 50 GGA AGC AAC CAC ATT GGA GAA TGG 30 Antisense 50 AGA ACC CCT GTG GTT TTG ACA 30

CD36 Mannose receptor Dectin-1

Please cite this article as: Cabanillas, B.J., et al., Leishmanicidal compounds and potent PPARγ activators from Renealmia thyrsoidea (Ruiz & Pav.) Poepp. & Endl.. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.010i

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promote the expression of Dectin-1 and MR, two macrophage pattern recognition receptors involved in Leishmania-macrophage interaction and whose transcriptional regulation requires PPARγ activation. As depicted in Fig. 3, both of them strongly induced the mRNA level of Dectin-1, especially compound 17 with a six-fold relative expression level compared to the control. Likewise, 17 and 18 strongly increased the expression of MR (two- to three-fold for 17 and 18, respectively). Our results indicated that compounds 17 and 18 are potent activators of PPARγ.

All these compounds were identified by the analysis of their spectroscopic properties and by comparison with published data Fig. 1. 3.2. Biological analyses The results of the inhibition against axenic amastigotes of Leishmania amazonensis are shown in Table 2. Three compounds 6, 7 and 11 showed moderate activity with IC50 values between 19.7 72.3 mM and 36.5 710.5 mM. The toxicity of compounds 6, 7 and 11 was also evaluated on murine peritoneal macrophages in order to establish a selectivity index, providing a very low LD50 value for 6 implying a high cytotoxicity/leishmanicidal activity ratio (411). Based on these results, an intracellular infected macrophages assay using 6 was performed. However, the compound displayed a very low activity with a reduction of infection only around 58% (Table 3). In addition, diarylheptanoids (14–18) were evaluated for their ability to activate PPARγ signaling pathways, through the measurement of CD36 mRNA level, a specific PPARγ target gene (Fig. 2). Diarylheptanoids 14–16 did not increase CD36 gene expression, whereas a twofold enhancement rate was observed with compounds 17 and 18, compared to the control without PPARγ ligand. Interestingly, these two compounds are more effective than rosiglitazone, a well-established PPARγ ligand. Then, we investigated whether diarylheptanoids 17 and 18

H

Previous phytochemical studies reported on different Renealmia species mainly resulted in the isolation of sesquiterpenes (Tchuendem et al., 1999; Kaplan et al., 2000), diterpenes (Zhou et al., 1997; Yang et al., 1999; Sekiguchi et al., 2001), anthocyanins (Gibaja-Oviedo, 1978), chalcones, flavanones (Gu et al., 2002) and cyclic diarylheptanoids (Sekiguchi et al., 2002). Although some of them displayed cytotoxic (Zhou et al., 1997) or antiplasmodial activities (Tchuendem et al., 1999), there is no report on their leishmanicidal properties. Eighteen compounds belonging to different classes of secondary metabolites were isolated from the leaves and the rhizomes of

OH

OH

H

O

4. Discussion

OH

H

HO

OH

3

2

1 OH

OH

OCH3

H

OH

H

H3CO

O OCH3 O

OH

H

HO

OH

OH 5

4

H

6

OCH3 H3CO

O

R1

O

8 R1 = OH 9 R1 = OCH3

R1

O

R2

O

O

R1

OH O 12 R1 = OH 13 R1 = OCH3

10 R1 = R2 = OH 11 R1 = R2 = OCH3

HO R1

OH

OCH3 H3CO

OH

7

OH

HO

OH

OH OH

OH O

14 R1 = H 15 R1 = OH

16

HO

OH

HO

OH

O

O 17

18

Fig. 1. Structures of compounds 1–18 isolated from Renealmia thyrsoidea.

Please cite this article as: Cabanillas, B.J., et al., Leishmanicidal compounds and potent PPARγ activators from Renealmia thyrsoidea (Ruiz & Pav.) Poepp. & Endl.. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.010i

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Table 2 Antileishmanial activity of compounds 1–18 and their cytotoxicity against peritoneal murine macrophages. Compound

CI50 (lM) Leishmania amazonensis axe. amas.

LD50 (lM) Cytotoxicity on macrophages

CARa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Amphotericin B

4209 4209 165.7 7 46.6 4195 118.7 7 35.2 36.5 710.5 19.7 7 2.3 4158 133.27 15.7 4159 23.17 5.3 4183 4163 4158 4150 4159 68.5 4169 0.157 0.01

NTb NT NT NT NT 4420 90.5 71.2 NT NT NT 79.6 75.7 NT NT NT NT NT NT NT 4.3

– – – – – 411 4.6 – – – 3.5 – – – – – – – 29

a b

Cytotoxicity on macrophage/antileishmanial activity ratio. NT: not tested.

Table 3 Activity in the intracellular macrophage-infected assay for compound 6. Compound

CI50 (lM)

% of RIa (MCb)

6 Amphotericin B

299 7 22.7 0.247 0.02

58.2 (336) 97.6 (1)

a

Reduction of infection. Maximum concentration (μM) that can be used without causing the death of macrophages. b

Fig. 2. CD36 mRNA expression in human monocytes-derived macrophages treated with rosiglitazone (Rosi) and the compounds 14–18.

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Renealmia thyrsoidea. Sesquiterpenes 1, 3, 4 and 5 were previously isolated from the fruits of Renealmia cincinnata (Tchuendem et al., 1999), while the classes of flavonols (8, 9), flavones (10, 11) and dibenzofurane (7) are reported here for the first time in the Renealmia genus. Diarylheptanoids are well-known and major secondary metabolites in the Zingiberaceae family, but their occurrence in the genus Renealmia was only described in the seeds of Renealmia exaltata (Sekiguchi et al., 2002). Their main representative and more studied member is curcumin, which displays various biological activities (see Gupta et al. (2011) and Esatbeyoglu et al. (2012) for reviews), including antileishmanial properties (see Haddad et al. (2011) for review). Renealmia thyrsoidea was thus investigated carefully to identify these kinds of compounds. As expected, five diarylheptanoids (14–18) were identified in the rhizome. From the leaves, compounds 6, 7 and 11 displayed a moderate leishmanicidal activity against Leishmania amazonensis axenic amastigotes, with IC50 values between 19.7 and 36.5 mM. We further examined the potency of the ( þ)-4β,10α-aromadendranediol (6) on Leishmania amazonensis infected macrophages, since it was not toxic on murine peritoneal macrophages. Compound 6 revealed to be inactive in this model, showing the relevance of this kind of assay in the screening for antileishmanial compounds. As a result, the antileishmanial activity of a leaf extract of Renealmia thrysoidea on our Leishmania amazonensis axenic amastigotes model (IC50 ¼19 μg/ml) could be explained by a synergy between compounds 6, 7 and/or 11, resulting in higher efficacy of the crude extracts when compared to isolated compounds. From the rhizomes, none of the isolated compounds exhibited leishmanicidal activity in our model. Flavone 12 and diarylheptanoid 17 were previously investigated for their antileishmanial properties against amastigotes stages. Compound 12 was claimed to be active against Leishmania donovani amastigotes (IC50 ¼ 5.0 mM, Tasdemir et al., 2006), and compound 17 was shown to be active against Leishmania mexicana amastigotes (IC50 ¼21.078 mM, Changtam et al., 2010). In our study, both compounds were inactive against Leishmania amazonensis axenic amastigotes. The discrepancies observed between our results and previous studies are justified if we consider that tests were performed with different Leishmania species, suggesting that 12 and 17 may be selective for a Leishmania species, as described for other compounds (Lima et al., 2004; de Morais-Teixeira et al., 2011). One could thus hypothesize that other minor active metabolites and/or synergistic effects of compounds that enhance the activity of these active principles, not identified yet, may be involved in the leishmanicidal activity of the crude rhizome extract (IC50 ¼10 μg/ml). It is now well established that synergism plays a key role in traditional medicines, as well as in infectious diseases treatment or cancer therapy, through e.g. enhancing bioavailability, activation of angiogenesis or immune system (Wagner and Ulrich-Merzenich, 2009; Rather et al., 2013). For example, curcumin, a very similar

Fig. 3. Dectin-1 and mannose receptor mRNA expression in human monocytes-derived macrophages treated with rosiglitazone (Rosi) and compounds 17–18.

Please cite this article as: Cabanillas, B.J., et al., Leishmanicidal compounds and potent PPARγ activators from Renealmia thyrsoidea (Ruiz & Pav.) Poepp. & Endl.. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.010i

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structure from our 14–18 isolated compounds, exerts a synergistic effect with 5-fluorouracil in colorectal cancer cells (Shakibaei et al. 2013, 2014). Indeed, curcumin has been shown to interact with multiple molecular targets such as NF-κB, Keap-1 or PPARγ (see Kunnumakkara et al. (2008), Gupta et al. (2011) and Esatbeyoglu et al. (2012) for reviews), strengthening the hypothesis of a synergic activity of these compounds. Regarding leishmaniasis, our team has demonstrated that two macrophage surface receptors, Dectin-1 and Mannose Receptor (MR), favored the macrophage response against Leishmania parasites (Lefèvre et al., 2013). Even if the mechanisms involved in the regulation of their expression remain unclear, we previously demonstrated that activation of PPARγ pathway is a key step in this host defense against pathogens (Coste et al., 2003; Galès et al., 2010). Those statements led us to investigate whether diarylheptanoids isolated from the rhizome of Renealmia thyrsoidea were able to activate PPARγ and thereby increase the expression of Dectin-1 and MR. To first assess the potency of diarylheptanoids (14–18) to activate PPARγ, we evaluated their ability to promote the expression of CD36, a PPARγ target gene. The results clearly indicated that compounds 17 and 18 enhanced the expression of CD36, whereas compounds 14–16 were inactive. Whether diarylheptanoids such as curcumin act as direct ligands of PPARγ or indirectly activate PPARγ remains controversial (Dong et al., 2011). In our study, lack of PPARγ activation with compounds 14–16 confirms that the electrophile α,β-unsaturated ketone moiety is involved in PPARγ activation. Our results are thus consistent with a covalent interaction through a Michael addition with sulfhydryl group of cysteines (Itoh et al., 2008; Waku et al., 2009; Festa et al., 2012) suggesting a direct binding with PPARγ. As the transcriptional regulation of MR and dectin-1 involves PPARγ, we then explored whether compounds 17 and 18 were able to induce their expression. As expected, both compounds 17 and 18 strongly increased the expression of MR and Dectin-1, indicating a possible effect on macrophage polarization since the expression of CD36, MR and Dectin-1 is characteristic of a M2-type phenotype (Lefèvre et al., 2013). These results cannot explain the activity of the rhizome extract, but rekindles the question of immunomodulators in phytotherapeutic or adaptogen approaches. Indeed, previous studies have revealed that curcumin might enhance innate immunity through up-regulation of CD36 surface expression on macrophages by activating Nrf2 signaling pathways, thus leading to the clearance of Plasmodium falciparum-parasitized erythrocytes (Olagnier et al., 2011; Mimche et al., 2011, 2012). It is worth mentioning that diarylheptanoids 17 and 18 displayed significant Nrf2 target genes expression enhancement in macrophages, such as heme-oxygenase 1 or glutathion reductase (data not shown). Furthermore, a Renealmia thyrsoidea rhizome extract displayed significant antimalarial properties (IC50 values of 6.8 71.5 mg/ml). Consequently, further investigations are required to confirm the prophagocytic activity of diarylheptanoids (17 and 18), and to explore their synergistic effects with antileishmanial or antimalarial compounds against Leishmania and malaria infections, respectively.

5. Conclusion In this study, 18 compounds were isolated from Renealmia thrysoidea. Among them, flavonols, flavones and dibenzofuranes were described here for the first time in the Renealmia genus. Our studies revealed moderate in vitro leishmanicidal activity for three compounds isolated from the leaves of Renealmia thyrsoidea (6, 7 and 11), while no leishmanicidal products were isolated from the rhizomes. On the other hand, two diarylheptanoids 17 and 18

bearing a Michael acceptor moiety exhibited interesting immunomodulatory properties towards macrophages. These findings were consistent with the traditional use of the aerial part of Renealmia thyrsoidea in the treatment of cutaneous leishmaniasis, and the use of a whole plant (leaves and rhizomes) preparation in case of fever or exhaustion. In that context, synergistic effects of potential antiparasitic compounds with immune-modifying derivatives are planned to be investigated in both leishmaniasis and malaria models, using a most relevant “host-targeted approach” with macrophages as primary effectors.

Acknowledgments The authors are thankful to DSF-IRD (BST) from France for the Q5 financial support to this work. We express our thanks to members of the Yanesha community who were willing to share their knowledge about medicinal plants with us.

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Please cite this article as: Cabanillas, B.J., et al., Leishmanicidal compounds and potent PPARγ activators from Renealmia thyrsoidea (Ruiz & Pav.) Poepp. & Endl.. Journal of Ethnopharmacology (2014), http://dx.doi.org/10.1016/j.jep.2014.09.010i