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Activity-guided isolation of antileishmanial compounds from Piper hispidum
Phytochemistry Letters 4 (2011) 363–366
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Activity-guided isolation of antileishmanial compounds from Piper hispidum Candy Ruiz a, Mohamed Haddad b,c, Joaquina Alban d, Genevie`ve Bourdy b,c, Ricardo Reategui a,1, Denis Castillo a, Michel Sauvain b,c, Eric Deharo b,c, Yannick Estevez b,c, Jorge Arevalo a, Rosario Rojas a,* a
Laboratorios de Investigacio´n y Desarrollo, Facultad de Ciencias y Filosofı´a, Universidad Peruana Cayetano Heredia, Lima, Peru IRD, UMR-152, Mission IRD Casilla, 18-1209 Lima, Peru c Universite´ de Toulouse, UPS, UMR152 (Laboratoire de Pharmacochimie et Pharmacologie pour le De´veloppement), 118, rte de Narbonne, F-31062 Toulouse Cedex 9, France d Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Av. Arenales 1256, Jesus Maria, Lima, Peru b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 June 2011 Received in revised form 20 July 2011 Accepted 3 August 2011 Available online 22 August 2011
The bioassay-guided purification of the ethanolic extract from the leaves of Piper hispidum led to the isolation of one new amide, N-2-(30 ,40 ,50 -trimethoxyphenyl)ethyl-2-hydroxybenzamide (1) as well as two known chalcones 20 -hydroxy-30 ,40 ,60 -trimethoxychalcone (2); 20 ,40 -dihydroxy-60 -methoxychalcone (cardamonin, 3) and one known flavanone, 5,7-dihydroxyflavanone (Pinocembrin, 4). Their structures were elucidated on the basis of spectroscopic data, including homo- and heteronuclear correlation NMR experiments (COSY, HSQC and HMBC) and comparison with data reported in the literature. The isolated compounds were tested against Leishmania amazonensis axenic amastigotes. The results showed that the known chalcone 2 exhibited the most potent antileishmanial activity with an IC50 of 0.8 mM (amphotericin B: IC50 = 0.2 mM) but was shown to exhibit mild cytotoxicity. ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Piper Amide Chalcone Flavanone Leishmaniasis
1. Introduction Leishmaniasis is a group of prevalent diseases caused by protozoan parasites belonging to the genus Leishmania extended over Africa, Asia, Europe, North and South America. An estimated 12 million people are infected worldwide in 88 countries with an annual incidence of about 2–3 million (Chava et al., 2005; Murray et al., 2005). The clinical manifestations may range from single cutaneous lesions to a fatal visceral form. Leishmaniasis is a serious problem for public health in the world, especially in tropical and subtropical regions where the parasites have developed resistance to current drugs. In the absence of effective vaccines, chemotherapy still plays a critical role in treating this disease. It relies on multiple parenteral injections with pentavalent antimonials that are considerably toxic (Guerin et al., 2002) and prone to induce resistance. Second-line drugs, such as amphotericin B, produce strong collateral effects, are costly for the majority of the populations in affected countries, and have restricted therapeutic spectra in different clinical forms of leishmaniasis (Davis et al., 2004). Recently, miltefosine has been shown to be active by oral
* Corresponding author. Tel.: +51 1 3190000x2705; fax: +51 1 3821762. E-mail address: [email protected] (R. Rojas). 1 Current address: AMRI, Bothell Research Center, 22215 26th Ave SE, Bothell, WA 98021, USA.
route against Bolivian mucosal leishmaniasis (Soto et al., 2007) but its efficacy remains to be determined against other types of leishmaniasis (Murray et al., 2005; Yardley et al., 2005; PerezVictoria et al., 2003). Therefore, the search for novel, effective, and safe drugs for the treatment of these diseases is essential for the control and prevention of leishmaniasis. One of the main opportunities is through the discovery of new antiparasitic agents from natural origins (Ioset, 2008). The genus Piper (Piperaceae), widely distributed in the tropical and subtropical regions of the world, is a pantropical group with nearly 2000 species, constituting an important element of mountain and lowland forests (Quijano-Abril et al., 2006). Phytochemical investigations of Piper species (Parmar et al., 1997) has reported the isolation of several classes of antiprotozoal compounds such as alkaloids (Rukachaisirikul et al., 2004), flavanones (Portet et al., 2007), chalcones and dihydrochalcones (Torres-Santos et al., 1999; Hermoso et al., 2003; Flores et al., 2007; Jenett-Siems et al., 1999), lignans (Cabanillas et al., 2010; Ma et al., 1991), neolignans (Luize et al., 2006). In South American traditional Pharmacopeias, the leaves of Piper hispidum Sw. and P. elongatum Vahl are used as poultices for healing wounds and to treat the symptoms of cutaneous leishmaniasis ‘‘Uta’’ (Estevez et al., 2007), and the leaves of P. aduncum L. are used for inflammation, and as antiseptic (Orjala et al., 1994). As part of our studies to uncover antileishmanial agents from the Peruvian biodiversity and provide scientific grounds for the
1874-3900/$ – see front matter ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.phytol.2011.08.001
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ethnomedicinal use of P. hispidum, we carried out an activityguided fractionation of P. hispidum against amastigote forms of Leishmania amazonensis in vitro. This extract exhibited interesting activity against axenic amastigotes of L. amazonensis, with an IC50 of 6.3 mg/ml. 2. Results and discussion The bioassay-guided fractionation of the ethanolic extract of P. hispidum leaves (IC50 = 6.3 mg/ml) against L. amazonensis axenic amastigotes yielded a methanolic fraction (IC50 = 5.1 mg/ml), from which a new compound (1) and three known compounds, including two chalcones (2, 3) and one flavanone (4) were isolated by column chromatography. The structures of compounds 1–4 were elucidated on the basis of spectroscopic data, including homo- and heteronuclear correlation NMR experiments (COSY, HSQC and HMBC) and comparison with data reported in the literature as 20 -hydroxy-30 ,40 ,60 -trimethoxychalcone (2), Cardamomin (20 ,40 -dihydroxy-60 -methoxychalcone) (3) and Pinocembrin (5,7-dihydroxyflavanone) (4), respectively (Vieira et al., 1980; Jaipetch et al., 1982; Kuroyanagi et al., 1983) (Fig. 1). Compound 1 (Fig. 1), was isolated as amorphous solid. The ESI mass spectrum (positive-ion mode) exhibited a quasimolecular ion peak at m/z 332 [M+H]+, indicating a molecular formula C18H21NO5. The UV absorption bands at 250 and 310 nm suggested the presence of N-benzoyltyramine skeleton. The IR absorption at 1641 cm1, the NMR signals at dC 169.9 (C-7), dC 161.6 (C-2), and dH 12.35 (OH-2) suggested the presence of a conjugated carbonyl engaged with a phenolic hydroxyl group in H-bonding. The 1H NMR spectrum indicated the presence of a 3,4,5-trimethoxyphenethylamino group supported by two MeO singlets at dH 3.84 (6H, s, MeO-30 ,50 ) and dH 3.86 (3H, s, MeO-40 ) which showed HSQC correlations with the carbons at d 56.5 and 60.9 respectively. This was confirmed by the observation of four CH2 protons at d 2.90 (2H, m, H-70 ) and 3.73 (2H, m, H-80 ), which correlated with the carbon signals at d 35.91 and 40.70 in the HSQC spectrum together with a NH signal at d 6.40 (1H, br s, NH). This was confirmed by comparison of the NMR data with known spectral data for structurally related compound, taiwanamide C (Chen et al., 2007). In addition, the presence of a 2-hydroxybenzoyl group could be supported by one OH signal at d 12.35 (1H, s, OH-2) and four
Fig. 2. Key HMBC correlations for compound 1.
mutually-coupling aromatic protons at d 6.80 (1H, m, H-4), 7.01 (1H, d, J 8.2 Hz, H-3), 7.22 (1H, dd, J 8.2 Hz, 1.6 Hz, H-6) and 7.41 (1H, m, H-5). This data was confirmed by comparison of the NMR data with compounds possessing a similar hydroxyphenyl moiety (Detsi et al., 2009) and supported by the HMBC correlations observed in the HMBC spectrum (Fig. 2). According to the above data, the structure of 1 was elucidated as N-2-(30 ,40 ,50 -trimethoxyphenyl)ethyl-2-hydroxybenzamide, a new natural compound. All the isolated compounds were tested for antileishmanial activity against axenic amastigotes of Leishmania amazonensis. Compound 2 was found to be the most active component of P. hispidum with an IC50 of 0.8 mM, followed by 3 with an IC50 of 8 mM. Both compounds were mildly toxic against peritoneal macrophages (IC50 = 1.6 and 18.2 mM, respectively). Compounds 1 and 4 were neither active against L. amazonensis amastigotes (IC50 > 40 mM) nor toxic to peritoneal macrophages (IC50 > 30 mM). 3. Experimental 3.1. General experimental procedures The UV spectra were recorded on Specord 205 spectrometer. IR spectroscopy was performed on a FT-IR Parabon Perkin Elmer spectrophotometer. The structures of the isolated compounds were identified by nuclear magnetic resonance (NMR; Bruker Avance 500 equipped with a TBI z-gradient 5 mm probe), 1H NMR (500 MHz), 13C NMR (125 MHz), and 2D NMR analysis in CDCl3;
Fig. 1. Chemical structures of 1–4.
C. Ruiz et al. / Phytochemistry Letters 4 (2011) 363–366
NMR experiments were performed at 298 K using standard pulse sequences. Chemical shifts (d) are given in ppm relative to TMS with coupling constant (J) reported in Hz. ESI-MS (positive and negative-ion mode) spectra were recorded on an ion trap LCQ Finnigan spectrometer. HRESI-MS spectra (positive and negativeion mode) were obtained on a Waters GCT Premier time-of-flight mass spectrometer. Column chromatography was carried out over silica gel (70–230 mesh, Merck). Fractions obtained from column chromatography were monitored by TLC (silica gel 60 F254, Merck) 3.2. Plant material Leaves of P. hispidum H.B.K. were collected in Alto Amazonas province, Loreto department, Peru. Voucher specimen (16180) was deposited at the National Herbarium of the San Marcos University in Lima, Peru (USM), and was identified by Joaquina Alba´n (Universidad Nacional Mayor de San Marcos). 3.3. Extraction and isolation Air-dried and powdered leaves of P. hispidum (1 kg) were crushed and extracted with 95% EtOH (20 l) for 6 days. After removing the solvent in vacuum, the extract (113 g) was partitioned into a CH2Cl2–H2O (1:1, v/v) solution, yielding the organic (74.6 g) and aqueous (1.9 g) extracts. After evaporation of the solvent under reduced pressure, the organic extract was partitioned into a Hexane-90% MeOH (1:1, v/v) solution, yielding the hexanic extract (28.5 g), the methanol extract (40.6 g) and a precipitate (4.1 g). A part of the methanol extract (15 g), which retains the biological activity, was subjected to column chromatography (60 mm 105 mm) on silica gel (300 g, 70–200 mm) and eluted with a gradient of n-Hexane/CH2Cl2/EtOAc (100/0/0–0/100/ 0–0/0/100) to afford 90 fractions (100 ml each). On the basis of their TLC profile, eight new fractions have been obtained (F1–F8). Fractions F4 (1.8 g) and F6 (2.0 g) displayed the best antileishmanial activity (IC50 = 7.5 and 7.9 mg/ml, respectively). Fraction F4 was further purified by column chromatography (27 mm 100 mm) on silica gel (35 g, 70–200 mm) using a mixture of petroleum ether/AcOEt increasing polarity as eluent (100/0–50/50) and then MeOH (100%) to give 7 fractions. The purification of the most potent fraction (F4.6, IC50 = 2.15 mg/ml) was further investigated. From F4.6 (94 mg), the compound 20 hydroxy-30 ,40 ,60 -trimethoxychalcone (2) was obtained after purification on an RP-18 (10 g, 40–60 mm) column chromatography (17 mm 100 mm) eluted with ACN/H2O (40/60–80/20). Fraction F6 (2 g) was submitted to column chromatography (27 mm 100 mm) on silica gel (35 g, 70–200 mm), eluted with a gradient of Et/EtOAc/CHCl3 (100/0/0–0/100/0–0/0/100) to afford 7 fractions. Fractions F6.6 (150 mg) proved to be the most active in the antileishmanial assay (IC50 = 2.2 mg/ml). Fraction F6.6 (150 mg) was further purified by silica gel (35 g, 70–200 mm) column chromatography (27 mm 100 mm) using a mixture of petroleum ether/AcOEt/MeOH (100/0/0–0/100/0–0/0/100) to give 5 fractions, with the higher antileishmanial activity concentrated on the fraction F6.6.3 (290 mg). This later was eluted on an RP-18 (30 g, 40–60 mm) column (27 mm 100 mm) chromatography eluted with different gradients of ACN/MeOH/H20 (0/0/100–0/100/ 0–100/0/0) to afford 4 fractions with the concentrated activity in F6.6.3.2 (100 mg). The new compound 1 (10 mg) was obtained after final purification on silica gel (10 g, 70–200 mm) column chromatography (17 mm 100 mm), eluted with a gradient of Et/ CHCl3/AcOEt (100/0/0–0/100/0–0/0/100). From F7 (4 g) compounds 3 (20 mg) and 4 (40 mg) were obtained after repeated purification on column chromatography (17 mm 100 mm) on silica gel (10 g, 70–200 mm), eluted with a gradient of Et/EtOAc/ MeOH (100/0/0–0/100/0–0/0/100).
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N-2-(30 ,40 ,50 -trimethoxyphenyl)ethyl-2-hydroxybenzamide (1): amorphous solid; IR bands (KBr): 3372 (NH), 1642 (C = O) cm1; 1H NMR: d 2.90 (2 H, m, H-70 ), 3.73 (2H, m, H-80 ), 3.84 (6H, s, MeO-30 ,50 ), 3.86 (3H, s, MeO-40 ), 6.40 (1H, br s, NH), 6.45 (2H, s, H20 , 60 ), 6.80 (1H, m, H-4), 7.01 (1H, d, J 8.2 Hz, H-3), 7.22 (1H, dd, J 8.2 Hz, 1.6 Hz, H-6), 7.41 (1H, m, H-5), 12.35 (1H, s, OH-2); 13C NMR: d 35.91 (C-70 ), 40.70 (C-80 ), 56.50 (MeO-30 , 50 ), 60.90 (MeO40 ), 105.59 (C-20 ,60 ), 114.2 (C-1), 118.6 (C-4), 118.7 (C-5), 125.07 (C6), 130.9 (C-10 ), 134.3 (C-3), 153.5 (C-30 ,50 ), 161.6 (C-2), 169.9 (C7); ESIMS m/z 332 [M+H]+. HRESIMS observed at m/z 332.1498 [M+H]+, calculated for C18H21NO5. 20 -hydroxy-30 ,40 ,60 -trimethoxychalcone (2): Negative ESIMS: m/z 313 [MH]; 1H and 13C NMR data are consistent with previously published data (Vieira et al., 1980). Cardamomin (20 ,40 -dihydroxy-60 -methoxychalcone) (3): yellow-orange crystals. Negative ESIMS: m/z 269 [MH]; 1H and 13C NMR data are consistent with previously published data (Jaipetch et al., 1982). Pinocembrin (5,7-dihydroxyflavanone) (4): white powder; Positive ESIMS: m/z 255 [MH]; 1H and 13C NMR data are consistent with previously published data (Kuroyanagi et al., 1983). 3.4. Bioassays Experiments were conducted on axenic amastigotes of Leishmania amazonensis (strain MHOM/BR/76/LTB-012) according to Estevez et al. (2007). Axenically grown amastigotes were maintained by weekly subpassages in medium for axenically grown amastigotes (extensively described by Sereno and Lemesre, 1997) and supplemented with 20% of foetal bovine serum at 32 8C with 5% CO2 in 25 cm2 tissue culture flasks. Cultures were initiated with 5 105 amastigotes or promastigotes/ml in 25 cm2 tissue culture flasks with 5 ml of medium. To determine the activity of the extracts, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) micromethod was used as previously described (Sereno and Lemesre, 1997). Briefly, 100 ml of axenically grown amastigotes or promastigotes, from late log phage of growth, were seeded in 96-well flat bottom microtiter plates. Extracts, dissolved in DMSO, were added at final concentrations ranging from 100 to 10 mg/ml. The final DMSO concentration was never greater than 0.1%. After 72 h of incubation, 10 ml of MTT (10 mg/ml) was added to each well and plates were further incubated for 4 h. The enzymatic reaction was then stopped with 100 ml of 50% isopropanol-10% sodium dodecyl sulfate and incubated for an additional 30 min under agitation at room temperature. Finally, the optical density (OD) was read at 570 nm with a 96-well scanner (Bio-Rad). All experiments were performed in triplicate, and standard deviations were calculated using Excel software. Percent growth inhibition of the parasite was calculated by the following formula:
% of inhibition ¼
OD control OD drugs 100 OD control
The concentration inhibiting 50% of the parasite growth (IC50) was calculated after evaluating percent growth inhibition at different concentrations. Reference compound was amphotericin B. Cytotoxicity of isolated compounds was determined on peritoneal macrophages prepared according to Sauvain et al. (1993). Non-inflammatory macrophages (105) were collected from 6-week-old male BALB/c mice. The adherent cells were incubated at 37 8C under 5% CO2 and appropriate dilutions of compounds were added. The contact between drugs and macrophages alone
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lasted 48 h at 37 8C under 5% CO2. The percentage of live macrophages was determined microscopically by means of the trypan blue dye exclusion test and the IC50 was determined by the same method as for amastigotes. Acknowledgements We thank Dr. Nicolas Fabre from the University of Toulouse, France, as well as the Department of Chemistry of the Pontificia Universidad Cato´lica del Peru´, for assistance in obtaining 1H, 13C NMR and MS spectra. References Cabanillas, B.J., Le Lamer, A.C., Castillo, D., Arevalo, J., Rojas, R., Odonne, G., Bourdy, G., Moukarzel, B., Sauvain, M., Fabre, N., 2010. Caffeic acid esters and lignans from Piper sanguineispicum. J. Nat. Prod. 73, 1884–1890. Chava, A.K., Chatterjee, M., Mandal, C., 2005. In: Yarema, K.J. (Ed.), Handbook of Carbohydrate Engineering. Taylor & Francis, Boca Raton, FL, pp. 71–98. Chen, I.S., Chen, Y.C., Liao, C.H., 2007. Amides with anti-platelet aggregation activity from Piper taiwanense. Fitoterapia 78, 414–419. Davis, A.J., Murray, H.W., Handman, E., 2004. Drug against leishmaniasis: a synergy of technology and partnerships. Trends Parasitol. 20, 73–76. Detsi, A., Majdalani, M., Kontogiorgis, C.A., Hadjipavlou-Litina, D., Kefalas, P., 2009. Natural and synthetic 20-hydroxy-chalcones and aurones: synthesis, characterization and evaluation of the antioxidant and soybean lipoxygenase inhibitory activity. Bioorg. Med. Chem. 17, 8073–8085. Estevez, Y., Castillo, D., Pisango, T., Arevalo, J., Rojas, R., Alban, J., Deharo, E., Bourdy, G., Sauvain, M., 2007. Evaluation of the leishmanicidal activity of plants used by Peruvian Chayahuita ethnic group. J. Ethnopharmacol. 114, 254–259. ˜ ero, J., Gime´nez, A., Bourdy, G., Corte´s-Selva, Flores, N., Cabrera, G., Jime´nez, I.A., Pin F., Bazzocchi, I.L., 2007. Leishmanicidal constituents from the leaves of Piper rusbyi. Planta Med. 73, 206–211. Guerin, P.J., Olliaro, P., Sundar, S., Boelaert, M., Croft, S.L., Desjeux, P., Wasunna, M.K., Brycerson, A.D., 2002. Visceral leishmaniasis: current status of control, diagnosis, and treatment, and a proposed research and development agenda. Lancet Infect. Dis. 2, 494–501. ˜ ero, J.E., Ravelo, A.G., Hermoso, A., Jime´nez, I.A., Mamani, Z.A., Bazzocchi, I.L., Pin Valladares, B., 2003. Antileishmanial activities of dihydrochalcones from Piper elongatum and synthetic related compounds. Structural requirements for activity. Bioorg. Med. Chem. 11, 3975–3980. Ioset, J.R., 2008. Natural products for neglected diseases: a review. Curr. Org. Chem. 12, 643–666. Jaipetch, T., Kanghae, S., Pancharoen, O., Patrick, V.A., Reutrakul, V., Tuntiwachwuttikul, P., White, A.H., 1982. Constitutes of Boesenbergia pandurata. Synthesis of (+)-boesenbergin A. Aust. J. Chem. 35, 351–361.
Jenett-Siems, K., Mockenhaupt, F.P., Bienzle, U., Gupta, M.P., Eich, E., 1999. In vitro antiplasmodial activity of Central American medicinal plants. Trop. Med. Int. Health 4, 611–615. Kuroyanagi, M., Noro, T., Fukushima, S., Aiyama, R., Ikuta, A., Itokawa, H., Morita, M., 1983. Chem. Pharm. Bull. 31, 1544–1550. Luize, P.S., Ueda-Nakamura, T., Filho, B.P., Corte´z, G.D., Nakamura, C.V., 2006. Activity of neolignans isolated from Piper regnellii (MIQ.) C.DC. var. pallescens (C.DC.) YUNCK against Trypanosoma cruzi. Biol. Pharm. Bull. 10, 2126–2130. Ma, Y., Han, G.Q., Li, C.L., Cheng, J.R., Arison, B.H., Hwang, S.B., 1991. Neolignans from Piper polysyphorum C.DC. Yao Xue Xue Bao 26, 345–350. Murray, H.W., Berman, J.D., Davies, C.R., Saravia, N.G., 2005. Advances in leishmaniasis. Lancet 366, 1561–1577. Orjala, J., Wright, A.D., Behrends, H., Folkers, G., Sticher, O., 1994. Cytotoxic and antibacterial dihydrochalcones from Piper aduncum. J. Nat. Prod. 57, 18–26. Parmar, V.S., Jain, S.C., Bisht, K.S., Jain, R., Taneja, P., Jha, A., Tyagi, O.D., Prasad, A.K., Wengel, J., Olsen, C.E., Boll, P.M., 1997. Phytochemistry of the genus Piper. Phytochemistry 46, 597–673. Perez-Victoria, F.J., Castanys, S., Gamarro, F., 2003. Leishmania donovani resistance to miltefosine involves a defective inward translocation of the drug. Antimicrob. Agents Chemother. 47, 2397–2403. Portet, B., Fabre, N., Roumy, V., Gornitzka, H., Bourdy, G., Chevalley, S., Sauvain, M., Valentin, A., Moulis, C., 2007. Activity-guided isolation of antiplasmodial dihydrochalcones and flavanones from Piper hostmannianum var. Berbicense. Phytochemistry 68, 1312–1320. Quijano-Abril, M.A., Callejas-Posadas, R., Miranda-Esquivel, D.R., 2006. Areas of endemism and distribution patterns for neotropical Piper species (Piperaceae). J. Biogeogr. 33, 1266–1278. Rukachaisirikul, T., Siriwattanakit, P., Sukcharoenphol, K., Wongvein, C., Ruttanaweang, P., Wongwattnavuch, P., Suksamrarn, A., 2004. Chemical constituents and bioactivity of Piper sarmentosum. J. Ethnopharmacol. 93, 173–176. Sauvain, M., Dedet, J.P., Kunesch, N., Poisson, J., Gayral, P., Gantier, J.C., Kunesch, G., 1993. In vitro and in vivo leishmanicidal activities of natural and synthethic quinoids. Phytother. Res. 7, 167–171. Sereno, D., Lemesre, J.-L., 1997. Axenically cultured amastigote forms as an in vitro model for investigation of antileishmanial agents. Antimicrob. Agents Chemother. 41, 972–976. Soto, J., Toledo, J., Valda, L., Balderrama, M., Rea, I., Parra, R., Ardiles, J., Soto, P., Gomez, A., Molleda, F., Fuentelsaz, C., Anders, G., Sindermann, H., Engel, J., Berman, J., 2007. Treatment of Bolivian mucosal leishmaniasis with miltefosine. Clin. Infect. Dis. 44, 350–356. Torres-Santos, E.C., Moreira, D.L., Kaplan, M.A.C., Meirelles, M.N., Rossi-Bergman, B., 1999. Selective effect of 20 ,60 -dihydroxy-40 -methoxychalcone isolated from Piper aduncum on Leishamnia amazonensis. Antimicrob. Agents Chemother. 43, 1234–1241. Vieira, P.C., De Alvarenga, M.A., Gottlieb, O.R., Gottlieb, H.E., 1980. 4-Hexadecenylphenol and flavonoids from Piper hispidum. Planta Med. 39, 153–156. Yardley, V., Croft, S.L., de Doncker, S., Dujardin, J.C., Koirala, S., Rijal, S., Miranda, C., Llanos-Cuentas, A., Chappuis, F., 2005. The sensitivity of clinical isolates of Leishmania from Peru and Nepal to miltefosine. Am. J. Trop. Med. Hyg. 73, 272–275.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Activity-guided isolation of antileishmanial compounds from Piper hispidum Candy Ruiz a, Mohamed Haddad b,c, Joaquina Alban d, Genevie`ve Bourdy b,c, Ricardo Reategui a,1, Denis Castillo a, Michel Sauvain b,c, Eric Deharo b,c, Yannick Estevez b,c, Jorge Arevalo a, Rosario Rojas a,* a
Laboratorios de Investigacio´n y Desarrollo, Facultad de Ciencias y Filosofı´a, Universidad Peruana Cayetano Heredia, Lima, Peru IRD, UMR-152, Mission IRD Casilla, 18-1209 Lima, Peru c Universite´ de Toulouse, UPS, UMR152 (Laboratoire de Pharmacochimie et Pharmacologie pour le De´veloppement), 118, rte de Narbonne, F-31062 Toulouse Cedex 9, France d Museo de Historia Natural, Universidad Nacional Mayor de San Marcos, Av. Arenales 1256, Jesus Maria, Lima, Peru b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 9 June 2011 Received in revised form 20 July 2011 Accepted 3 August 2011 Available online 22 August 2011
The bioassay-guided purification of the ethanolic extract from the leaves of Piper hispidum led to the isolation of one new amide, N-2-(30 ,40 ,50 -trimethoxyphenyl)ethyl-2-hydroxybenzamide (1) as well as two known chalcones 20 -hydroxy-30 ,40 ,60 -trimethoxychalcone (2); 20 ,40 -dihydroxy-60 -methoxychalcone (cardamonin, 3) and one known flavanone, 5,7-dihydroxyflavanone (Pinocembrin, 4). Their structures were elucidated on the basis of spectroscopic data, including homo- and heteronuclear correlation NMR experiments (COSY, HSQC and HMBC) and comparison with data reported in the literature. The isolated compounds were tested against Leishmania amazonensis axenic amastigotes. The results showed that the known chalcone 2 exhibited the most potent antileishmanial activity with an IC50 of 0.8 mM (amphotericin B: IC50 = 0.2 mM) but was shown to exhibit mild cytotoxicity. ß 2011 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Piper Amide Chalcone Flavanone Leishmaniasis
1. Introduction Leishmaniasis is a group of prevalent diseases caused by protozoan parasites belonging to the genus Leishmania extended over Africa, Asia, Europe, North and South America. An estimated 12 million people are infected worldwide in 88 countries with an annual incidence of about 2–3 million (Chava et al., 2005; Murray et al., 2005). The clinical manifestations may range from single cutaneous lesions to a fatal visceral form. Leishmaniasis is a serious problem for public health in the world, especially in tropical and subtropical regions where the parasites have developed resistance to current drugs. In the absence of effective vaccines, chemotherapy still plays a critical role in treating this disease. It relies on multiple parenteral injections with pentavalent antimonials that are considerably toxic (Guerin et al., 2002) and prone to induce resistance. Second-line drugs, such as amphotericin B, produce strong collateral effects, are costly for the majority of the populations in affected countries, and have restricted therapeutic spectra in different clinical forms of leishmaniasis (Davis et al., 2004). Recently, miltefosine has been shown to be active by oral
* Corresponding author. Tel.: +51 1 3190000x2705; fax: +51 1 3821762. E-mail address: [email protected] (R. Rojas). 1 Current address: AMRI, Bothell Research Center, 22215 26th Ave SE, Bothell, WA 98021, USA.
route against Bolivian mucosal leishmaniasis (Soto et al., 2007) but its efficacy remains to be determined against other types of leishmaniasis (Murray et al., 2005; Yardley et al., 2005; PerezVictoria et al., 2003). Therefore, the search for novel, effective, and safe drugs for the treatment of these diseases is essential for the control and prevention of leishmaniasis. One of the main opportunities is through the discovery of new antiparasitic agents from natural origins (Ioset, 2008). The genus Piper (Piperaceae), widely distributed in the tropical and subtropical regions of the world, is a pantropical group with nearly 2000 species, constituting an important element of mountain and lowland forests (Quijano-Abril et al., 2006). Phytochemical investigations of Piper species (Parmar et al., 1997) has reported the isolation of several classes of antiprotozoal compounds such as alkaloids (Rukachaisirikul et al., 2004), flavanones (Portet et al., 2007), chalcones and dihydrochalcones (Torres-Santos et al., 1999; Hermoso et al., 2003; Flores et al., 2007; Jenett-Siems et al., 1999), lignans (Cabanillas et al., 2010; Ma et al., 1991), neolignans (Luize et al., 2006). In South American traditional Pharmacopeias, the leaves of Piper hispidum Sw. and P. elongatum Vahl are used as poultices for healing wounds and to treat the symptoms of cutaneous leishmaniasis ‘‘Uta’’ (Estevez et al., 2007), and the leaves of P. aduncum L. are used for inflammation, and as antiseptic (Orjala et al., 1994). As part of our studies to uncover antileishmanial agents from the Peruvian biodiversity and provide scientific grounds for the
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ethnomedicinal use of P. hispidum, we carried out an activityguided fractionation of P. hispidum against amastigote forms of Leishmania amazonensis in vitro. This extract exhibited interesting activity against axenic amastigotes of L. amazonensis, with an IC50 of 6.3 mg/ml. 2. Results and discussion The bioassay-guided fractionation of the ethanolic extract of P. hispidum leaves (IC50 = 6.3 mg/ml) against L. amazonensis axenic amastigotes yielded a methanolic fraction (IC50 = 5.1 mg/ml), from which a new compound (1) and three known compounds, including two chalcones (2, 3) and one flavanone (4) were isolated by column chromatography. The structures of compounds 1–4 were elucidated on the basis of spectroscopic data, including homo- and heteronuclear correlation NMR experiments (COSY, HSQC and HMBC) and comparison with data reported in the literature as 20 -hydroxy-30 ,40 ,60 -trimethoxychalcone (2), Cardamomin (20 ,40 -dihydroxy-60 -methoxychalcone) (3) and Pinocembrin (5,7-dihydroxyflavanone) (4), respectively (Vieira et al., 1980; Jaipetch et al., 1982; Kuroyanagi et al., 1983) (Fig. 1). Compound 1 (Fig. 1), was isolated as amorphous solid. The ESI mass spectrum (positive-ion mode) exhibited a quasimolecular ion peak at m/z 332 [M+H]+, indicating a molecular formula C18H21NO5. The UV absorption bands at 250 and 310 nm suggested the presence of N-benzoyltyramine skeleton. The IR absorption at 1641 cm1, the NMR signals at dC 169.9 (C-7), dC 161.6 (C-2), and dH 12.35 (OH-2) suggested the presence of a conjugated carbonyl engaged with a phenolic hydroxyl group in H-bonding. The 1H NMR spectrum indicated the presence of a 3,4,5-trimethoxyphenethylamino group supported by two MeO singlets at dH 3.84 (6H, s, MeO-30 ,50 ) and dH 3.86 (3H, s, MeO-40 ) which showed HSQC correlations with the carbons at d 56.5 and 60.9 respectively. This was confirmed by the observation of four CH2 protons at d 2.90 (2H, m, H-70 ) and 3.73 (2H, m, H-80 ), which correlated with the carbon signals at d 35.91 and 40.70 in the HSQC spectrum together with a NH signal at d 6.40 (1H, br s, NH). This was confirmed by comparison of the NMR data with known spectral data for structurally related compound, taiwanamide C (Chen et al., 2007). In addition, the presence of a 2-hydroxybenzoyl group could be supported by one OH signal at d 12.35 (1H, s, OH-2) and four
Fig. 2. Key HMBC correlations for compound 1.
mutually-coupling aromatic protons at d 6.80 (1H, m, H-4), 7.01 (1H, d, J 8.2 Hz, H-3), 7.22 (1H, dd, J 8.2 Hz, 1.6 Hz, H-6) and 7.41 (1H, m, H-5). This data was confirmed by comparison of the NMR data with compounds possessing a similar hydroxyphenyl moiety (Detsi et al., 2009) and supported by the HMBC correlations observed in the HMBC spectrum (Fig. 2). According to the above data, the structure of 1 was elucidated as N-2-(30 ,40 ,50 -trimethoxyphenyl)ethyl-2-hydroxybenzamide, a new natural compound. All the isolated compounds were tested for antileishmanial activity against axenic amastigotes of Leishmania amazonensis. Compound 2 was found to be the most active component of P. hispidum with an IC50 of 0.8 mM, followed by 3 with an IC50 of 8 mM. Both compounds were mildly toxic against peritoneal macrophages (IC50 = 1.6 and 18.2 mM, respectively). Compounds 1 and 4 were neither active against L. amazonensis amastigotes (IC50 > 40 mM) nor toxic to peritoneal macrophages (IC50 > 30 mM). 3. Experimental 3.1. General experimental procedures The UV spectra were recorded on Specord 205 spectrometer. IR spectroscopy was performed on a FT-IR Parabon Perkin Elmer spectrophotometer. The structures of the isolated compounds were identified by nuclear magnetic resonance (NMR; Bruker Avance 500 equipped with a TBI z-gradient 5 mm probe), 1H NMR (500 MHz), 13C NMR (125 MHz), and 2D NMR analysis in CDCl3;
Fig. 1. Chemical structures of 1–4.
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NMR experiments were performed at 298 K using standard pulse sequences. Chemical shifts (d) are given in ppm relative to TMS with coupling constant (J) reported in Hz. ESI-MS (positive and negative-ion mode) spectra were recorded on an ion trap LCQ Finnigan spectrometer. HRESI-MS spectra (positive and negativeion mode) were obtained on a Waters GCT Premier time-of-flight mass spectrometer. Column chromatography was carried out over silica gel (70–230 mesh, Merck). Fractions obtained from column chromatography were monitored by TLC (silica gel 60 F254, Merck) 3.2. Plant material Leaves of P. hispidum H.B.K. were collected in Alto Amazonas province, Loreto department, Peru. Voucher specimen (16180) was deposited at the National Herbarium of the San Marcos University in Lima, Peru (USM), and was identified by Joaquina Alba´n (Universidad Nacional Mayor de San Marcos). 3.3. Extraction and isolation Air-dried and powdered leaves of P. hispidum (1 kg) were crushed and extracted with 95% EtOH (20 l) for 6 days. After removing the solvent in vacuum, the extract (113 g) was partitioned into a CH2Cl2–H2O (1:1, v/v) solution, yielding the organic (74.6 g) and aqueous (1.9 g) extracts. After evaporation of the solvent under reduced pressure, the organic extract was partitioned into a Hexane-90% MeOH (1:1, v/v) solution, yielding the hexanic extract (28.5 g), the methanol extract (40.6 g) and a precipitate (4.1 g). A part of the methanol extract (15 g), which retains the biological activity, was subjected to column chromatography (60 mm 105 mm) on silica gel (300 g, 70–200 mm) and eluted with a gradient of n-Hexane/CH2Cl2/EtOAc (100/0/0–0/100/ 0–0/0/100) to afford 90 fractions (100 ml each). On the basis of their TLC profile, eight new fractions have been obtained (F1–F8). Fractions F4 (1.8 g) and F6 (2.0 g) displayed the best antileishmanial activity (IC50 = 7.5 and 7.9 mg/ml, respectively). Fraction F4 was further purified by column chromatography (27 mm 100 mm) on silica gel (35 g, 70–200 mm) using a mixture of petroleum ether/AcOEt increasing polarity as eluent (100/0–50/50) and then MeOH (100%) to give 7 fractions. The purification of the most potent fraction (F4.6, IC50 = 2.15 mg/ml) was further investigated. From F4.6 (94 mg), the compound 20 hydroxy-30 ,40 ,60 -trimethoxychalcone (2) was obtained after purification on an RP-18 (10 g, 40–60 mm) column chromatography (17 mm 100 mm) eluted with ACN/H2O (40/60–80/20). Fraction F6 (2 g) was submitted to column chromatography (27 mm 100 mm) on silica gel (35 g, 70–200 mm), eluted with a gradient of Et/EtOAc/CHCl3 (100/0/0–0/100/0–0/0/100) to afford 7 fractions. Fractions F6.6 (150 mg) proved to be the most active in the antileishmanial assay (IC50 = 2.2 mg/ml). Fraction F6.6 (150 mg) was further purified by silica gel (35 g, 70–200 mm) column chromatography (27 mm 100 mm) using a mixture of petroleum ether/AcOEt/MeOH (100/0/0–0/100/0–0/0/100) to give 5 fractions, with the higher antileishmanial activity concentrated on the fraction F6.6.3 (290 mg). This later was eluted on an RP-18 (30 g, 40–60 mm) column (27 mm 100 mm) chromatography eluted with different gradients of ACN/MeOH/H20 (0/0/100–0/100/ 0–100/0/0) to afford 4 fractions with the concentrated activity in F6.6.3.2 (100 mg). The new compound 1 (10 mg) was obtained after final purification on silica gel (10 g, 70–200 mm) column chromatography (17 mm 100 mm), eluted with a gradient of Et/ CHCl3/AcOEt (100/0/0–0/100/0–0/0/100). From F7 (4 g) compounds 3 (20 mg) and 4 (40 mg) were obtained after repeated purification on column chromatography (17 mm 100 mm) on silica gel (10 g, 70–200 mm), eluted with a gradient of Et/EtOAc/ MeOH (100/0/0–0/100/0–0/0/100).
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N-2-(30 ,40 ,50 -trimethoxyphenyl)ethyl-2-hydroxybenzamide (1): amorphous solid; IR bands (KBr): 3372 (NH), 1642 (C = O) cm1; 1H NMR: d 2.90 (2 H, m, H-70 ), 3.73 (2H, m, H-80 ), 3.84 (6H, s, MeO-30 ,50 ), 3.86 (3H, s, MeO-40 ), 6.40 (1H, br s, NH), 6.45 (2H, s, H20 , 60 ), 6.80 (1H, m, H-4), 7.01 (1H, d, J 8.2 Hz, H-3), 7.22 (1H, dd, J 8.2 Hz, 1.6 Hz, H-6), 7.41 (1H, m, H-5), 12.35 (1H, s, OH-2); 13C NMR: d 35.91 (C-70 ), 40.70 (C-80 ), 56.50 (MeO-30 , 50 ), 60.90 (MeO40 ), 105.59 (C-20 ,60 ), 114.2 (C-1), 118.6 (C-4), 118.7 (C-5), 125.07 (C6), 130.9 (C-10 ), 134.3 (C-3), 153.5 (C-30 ,50 ), 161.6 (C-2), 169.9 (C7); ESIMS m/z 332 [M+H]+. HRESIMS observed at m/z 332.1498 [M+H]+, calculated for C18H21NO5. 20 -hydroxy-30 ,40 ,60 -trimethoxychalcone (2): Negative ESIMS: m/z 313 [MH]; 1H and 13C NMR data are consistent with previously published data (Vieira et al., 1980). Cardamomin (20 ,40 -dihydroxy-60 -methoxychalcone) (3): yellow-orange crystals. Negative ESIMS: m/z 269 [MH]; 1H and 13C NMR data are consistent with previously published data (Jaipetch et al., 1982). Pinocembrin (5,7-dihydroxyflavanone) (4): white powder; Positive ESIMS: m/z 255 [MH]; 1H and 13C NMR data are consistent with previously published data (Kuroyanagi et al., 1983). 3.4. Bioassays Experiments were conducted on axenic amastigotes of Leishmania amazonensis (strain MHOM/BR/76/LTB-012) according to Estevez et al. (2007). Axenically grown amastigotes were maintained by weekly subpassages in medium for axenically grown amastigotes (extensively described by Sereno and Lemesre, 1997) and supplemented with 20% of foetal bovine serum at 32 8C with 5% CO2 in 25 cm2 tissue culture flasks. Cultures were initiated with 5 105 amastigotes or promastigotes/ml in 25 cm2 tissue culture flasks with 5 ml of medium. To determine the activity of the extracts, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) micromethod was used as previously described (Sereno and Lemesre, 1997). Briefly, 100 ml of axenically grown amastigotes or promastigotes, from late log phage of growth, were seeded in 96-well flat bottom microtiter plates. Extracts, dissolved in DMSO, were added at final concentrations ranging from 100 to 10 mg/ml. The final DMSO concentration was never greater than 0.1%. After 72 h of incubation, 10 ml of MTT (10 mg/ml) was added to each well and plates were further incubated for 4 h. The enzymatic reaction was then stopped with 100 ml of 50% isopropanol-10% sodium dodecyl sulfate and incubated for an additional 30 min under agitation at room temperature. Finally, the optical density (OD) was read at 570 nm with a 96-well scanner (Bio-Rad). All experiments were performed in triplicate, and standard deviations were calculated using Excel software. Percent growth inhibition of the parasite was calculated by the following formula:
% of inhibition ¼
OD control OD drugs 100 OD control
The concentration inhibiting 50% of the parasite growth (IC50) was calculated after evaluating percent growth inhibition at different concentrations. Reference compound was amphotericin B. Cytotoxicity of isolated compounds was determined on peritoneal macrophages prepared according to Sauvain et al. (1993). Non-inflammatory macrophages (105) were collected from 6-week-old male BALB/c mice. The adherent cells were incubated at 37 8C under 5% CO2 and appropriate dilutions of compounds were added. The contact between drugs and macrophages alone
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lasted 48 h at 37 8C under 5% CO2. The percentage of live macrophages was determined microscopically by means of the trypan blue dye exclusion test and the IC50 was determined by the same method as for amastigotes. Acknowledgements We thank Dr. Nicolas Fabre from the University of Toulouse, France, as well as the Department of Chemistry of the Pontificia Universidad Cato´lica del Peru´, for assistance in obtaining 1H, 13C NMR and MS spectra. References Cabanillas, B.J., Le Lamer, A.C., Castillo, D., Arevalo, J., Rojas, R., Odonne, G., Bourdy, G., Moukarzel, B., Sauvain, M., Fabre, N., 2010. Caffeic acid esters and lignans from Piper sanguineispicum. J. Nat. Prod. 73, 1884–1890. Chava, A.K., Chatterjee, M., Mandal, C., 2005. In: Yarema, K.J. (Ed.), Handbook of Carbohydrate Engineering. Taylor & Francis, Boca Raton, FL, pp. 71–98. Chen, I.S., Chen, Y.C., Liao, C.H., 2007. Amides with anti-platelet aggregation activity from Piper taiwanense. Fitoterapia 78, 414–419. Davis, A.J., Murray, H.W., Handman, E., 2004. Drug against leishmaniasis: a synergy of technology and partnerships. Trends Parasitol. 20, 73–76. Detsi, A., Majdalani, M., Kontogiorgis, C.A., Hadjipavlou-Litina, D., Kefalas, P., 2009. Natural and synthetic 20-hydroxy-chalcones and aurones: synthesis, characterization and evaluation of the antioxidant and soybean lipoxygenase inhibitory activity. Bioorg. Med. Chem. 17, 8073–8085. Estevez, Y., Castillo, D., Pisango, T., Arevalo, J., Rojas, R., Alban, J., Deharo, E., Bourdy, G., Sauvain, M., 2007. Evaluation of the leishmanicidal activity of plants used by Peruvian Chayahuita ethnic group. J. Ethnopharmacol. 114, 254–259. ˜ ero, J., Gime´nez, A., Bourdy, G., Corte´s-Selva, Flores, N., Cabrera, G., Jime´nez, I.A., Pin F., Bazzocchi, I.L., 2007. Leishmanicidal constituents from the leaves of Piper rusbyi. Planta Med. 73, 206–211. Guerin, P.J., Olliaro, P., Sundar, S., Boelaert, M., Croft, S.L., Desjeux, P., Wasunna, M.K., Brycerson, A.D., 2002. Visceral leishmaniasis: current status of control, diagnosis, and treatment, and a proposed research and development agenda. Lancet Infect. Dis. 2, 494–501. ˜ ero, J.E., Ravelo, A.G., Hermoso, A., Jime´nez, I.A., Mamani, Z.A., Bazzocchi, I.L., Pin Valladares, B., 2003. Antileishmanial activities of dihydrochalcones from Piper elongatum and synthetic related compounds. Structural requirements for activity. Bioorg. Med. Chem. 11, 3975–3980. Ioset, J.R., 2008. Natural products for neglected diseases: a review. Curr. Org. Chem. 12, 643–666. Jaipetch, T., Kanghae, S., Pancharoen, O., Patrick, V.A., Reutrakul, V., Tuntiwachwuttikul, P., White, A.H., 1982. Constitutes of Boesenbergia pandurata. Synthesis of (+)-boesenbergin A. Aust. J. Chem. 35, 351–361.
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