A New Furanoheliangolide Sesquiterpene Lactone from Calea pinnatifida ( R . Br . ) Less . ( Asteraceae ) and Evaluation of Its Trypanocidal and Leishmanicidal Activities

Calea pinnatifida (R. Br.) Less. is commonly known in Brazil as “cipó-cruz”, “quebra-tudo” or “aruca”. This species is employed in folk medicine as giardicidal, amoebicidal and to treat digestive disorders. The present paper describes the isolation and structure determination of a new furanoheliangolide sesquiterpene lactone named 11,13-dihydroxy-calaxin, in addition to seven known compounds: ethyl caffeate, vanillin, 12-hydroxy-encecalin, phytol, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid from the ethanol extract of C. pinnatifida leaves. Additionally, 11,13-dihydroxy-calaxin, vanillin, 12-hydroxy-encecalin, phytol, 3,4-di-O-caffeoylquinic acid, 4,5-di-O-caffeoylquinic acid and the mixture of 3,4-di-Ocaffeoylquinic, 3,5-di-O-caffeoylquinic and 4,5-di-O-caffeoylquinic acids were assayed against the amastigote forms of Leishmania amazonensis and Trypanosoma cruzi, and the sesquiterpene lactone 11,13-dihydroxy-calaxin exhibited a promising trypanocidal and leishmanicidal activity, displaying IC50 values of 5.27 and 8.30 μM, respectively.


Introduction
Leishmaniasis and Chagas' disease constitute serious health care problems, mostly in Sub-Saharan Africa, Latin America and Middle East Asia.These parasitic diseases are caused by kinetoplastid protozoan of the genus Leishmania and Trypanosoma, respectively, and are responsible for substantial global morbidity, mortality, economic adversity and huge health care costs. 1,2The available chemotherapy for the treatment of these illnesses presents important limitations, including partial efficacy, toxicity, side effects, long-term therapies and high number of resistance cases, resulting in treatment failure. 3Thus, there is an urgent demand for the discovery of new antiprotozoal compounds and/or the development of novel therapeutic strategies which are safer, effective, accessible and less toxic.In this context, the natural products from plants, in particular, represent a remarkable source to furnish new bioactive substances. 4,5alea pinnatifida (R. Br.) Less.[9] To the best of our knowledge, few chemical and biological works have been performed with this species.Previous phytochemical studies on aerial parts and leaves from C. pinnatifida led to the isolation and identification of one polyacetylene, one sesquiterpene lactone, fatty acid esters, steroids, benzoic acid derivatives 10,11 and chromenes. 6urthermore, it has also been reported antiproliferative and leishmanicidal properties for some of its extracts (dichloromethane and ethanol) or chemical constituents (sesquiterpene lactones and chromenes). 6,12hus, the main goals of the present study were to explore the chemical composition from the ethanol extract of the leaves of C. pinnatifida, as well as to evaluate the in vitro trypanocidal and leishmanicidal activities of the isolated compounds against intracellular forms (amastigotes) of Trypanosoma cruzi and Leishmania amazonensis.Polartronic E polarimeter.All NMR analysis ( 1 H, 13 C, correlation spectroscopy (COSY), nuclear Overhauser spectroscopy (NOESY), heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC)) were carried out employing Bruker Fourier 300 UltraShield, Bruker AVANCE-400 and/or Ascend 600 spectrometers, operating at 300, 400 and 600 MHz for 1 H; and 75, 100 and 150 MHz for 13 C, respectively.CDCl 3 , acetone-d 6 or methanol-d 4 with TMS as internal standard (0.00 ppm) were used as solvents for acquisition of the NMR spectra.Chemical shifts (d) were given in ppm and coupling constants (J) were expressed in hertz (Hz).High-resolution electrospray ionization mass spectra (HRESIMS) were measured on an Exactive TM Plus Orbitrap mass spectrometer (Thermo Fisher Scientific) and/or Xevo ® G2-XS QTof mass spectrometer (Waters).Preparative high-performance liquid chromatography (HPLC) was performed on a Shimadzu HPLC system (Kyoto, Japan) equipped with two LC-10AD pumps, SCL-10ADVP system controller, SPD-10AV UV detector and manual injection system, using a C18 column Luna type (10 μm, 250 × 10 mm, Phenomenex ® ).Medium pressure liquid chromatographic (MPLC) separation was carried out using an FMI fluid metering lab pump, model QSY (Fluid Metering Inc., Syosset, NY).Silica gel (230-400 mesh) used for column chromatography and vacuum liquid chromatography was obtained from Vetec, Brazil; analytical TLC and silica gel RP-18 (240-400 mesh) were purchased from Silicycle; and Sephadex LH-20 was obtained from Tedia Brazil, Brazil.

Collection of plant material
C. pinnatifida (R. Br.) Less.leaves were collected at the "Costa da Lagoa", Florianópolis, Santa Catarina, Brazil, in June 2013.Botanical identification and authentication of the plant material were performed by PhD John F. Pruski, New York Botanical Garden, and specimen samples are deposited in the Missouri Botanical Garden Herbarium, St. Louis, Missouri, USA (No: MO-2383318) and in the Rio de Janeiro Botanical Garden Herbarium, Rio de Janeiro, Brazil (No: RB00906349).

Extraction and isolation of the chemical constituents
Fresh leaves from C. pinnatifida (2.6 kg) were cut into small irregular pieces with a scissors and submitted to maceration with ethanol 92% for 15 days at room temperature (ca. 25 ºC) until exhaustion (solvent was renewed four times).The solvent was concentrated using a rotary evaporator at 40 ºC under reduced pressure to afford 142.0 g of a dark green crude extract.Crude extract was solubilized in a minimum volume of H 2 O and sequentially partitioned with hexane (5 × 500 mL), dichloromethane (4 × 500 mL), and ethyl acetate (3 × 500 mL), to yield hexane (68.5 g), dichloromethane (7.6 g) and ethyl acetate (5.9 g) phases, as well as a residual aqueous fraction that was lyophilized to give a hygroscopic solid (60.0 g).
To perform the screening of the leishmanicidal activity against intracellular amastigotes of L. amazonensis, THP-1 cells (3.0 × 10 4 per well) were cultivated in 96-well plates using the RPMI-1640 medium supplemented as described previously, treated with 100 ng mL -1 of phorbol 12-myristate 13-acetate (PMA) at 37 °C in a 5% CO 2 for 72 h, to allow THP-1 cells differentiation into non-dividing macrophages. 14our days culture promastigotes (4.0 × 10 6 parasites per mL) were washed with phosphate buffered saline (PBS), pH 7.4, and incubated in RPMI-1640 medium supplemented with 10% human AB + serum heatinactivated at 34 °C for 1 h to parasite opsonization.THP-1 cells were incubated with a parasite:cell ratio of 10:1 for 3 h at 34 °C (CO 2 5%).After this period, non-adherent parasites were removed by one wash with PBS and infected cells were incubated with 180 μL of complete supplemented RPMI-1640 medium for another 24 h to allow the transformation of promastigotes into intracellular amastigotes.The β-galactosidase Trypanosoma cruzi, Tulahuen strain, 15 was kindly provided by the Laboratório de Parasitologia Molecular e Celular, Centro de Pesquisas René Rachou, FIOCRUZ, Belo Horizonte, Brazil.Culturederived trypomastigotes raised from infected L929 cell line were used to infect differentiated THP-1 (4.0 × 10 4 cells per well) in 96-well microplates in a parasite:cell ratio of 3:1 and incubated overnight at 37 ºC with 5% CO 2 .The medium containing non-internalized parasites was removed and replaced with 180 μL of fresh medium.
Compounds 1, 3-5, 6, 8 and the mixture of 6 + 7 + 8 were dissolved in DMSO 1% at concentrations ranging from 50 to 1.56 μM.Infected cell layer was treated by addition of 20 μL of each sample followed by incubation at 34 °C with 5% CO 2 for 48 h.After treatment, cells were carefully washed with PBS and incubated at 37 °C for 16 h with 250 μL of chlorophenol red-β-D-galactopyranoside (CPRG) (Sigma-Aldrich Co., St. Louis, MO, USA) at 100 μM and Nonidet P-40 0.1% (NP-40) (Amresco Inc, Solon, Ohio, USA).All assays were performed in triplicate.Optical density was read at 570/630 nm in an Infinite M200 TECAN, Austria.The concentration of each sample that reduced parasite viability by 50% when compared to untreated control (IC 50 ) was estimated by non-linear regression of concentration-response curves.Benznidazole (Sigma) and amphotericin B (Bristol-Myers, Squibb) were utilized as positive control groups for trypanocidal and leishmanicidal assays, respectively, and DMSO 1% was used as negative control group.

Cell toxicity assay
THP-1 cells were seeded (6.0 × 10 4 per well) in 96-well microplates and differentiated with 100 ng mL -1 of PMA.After 72 h of incubation at 37 ºC in a humidified 5% CO 2 atmosphere, compounds at concentration varying between 500 and 15.6 μM or DMSO 1% (negative control) were added and the cells were incubated again for 72 h at 37 ºC (CO 2 5%).The cell viability was measured by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method (MTT) based on tetrazolium salt reduction by mitochondrial dehydrogenases, and the optical densities were read at 540 nm in a TECAN Infinite M200 microplate reader immediately after the dissolution of formazan crystals with DMSO.The CC 50 value was defined as the concentration of each compound that reduced the absorbance of treated cells by 50% when compared with the cell control. 16
Compound 1 was obtained from the dichloromethane fraction as a white solid with melting point = 144.5-146.0ºC.Further, this compound appeared as a dark brown spot on TLC after spraying with anisaldehyde-sulphuric acid and heating at 100 ºC for few minutes.Its molecular formula was determined by HRESIMS in both positive and negative ionization modes.The HRESIMS spectrum in positive mode exhibited a molecular ion peak at m/z 379. ) fragment ions, due to the elimination of CO 2 and H 2 O, respectively.A fragmentation mechanism for the sesquiterpene lactone 1 is proposed in the Figure 3.
The relative configurations of stereogenic centers in compound 1 were deduced on the basis in the coupling constant values which were correlated with the dihedral angles obtained from Dreiding models and application of the Karplus curve.The relative cis-disposition of C(4,5) double bond associated with the coupling constant value recorded between H-6/H-7 (J 6,7 4.2 Hz) indicated a different arrangement for H-6 and H-7.Assuming that H-7 is a-oriented, as generally occurs in sesquiterpene lactones isolated from higher plants, [29][30][31] it should be concluded a β-orientation for H-6.Similarly, the small coupling constant observed between H-7/H-8 (J 7,8 2.2 Hz) suggested a β-orientation for the methacrylate side chain located at C-8 and a-orientation for the oxymethine H-8.The signal corresponding to the H-7 hydrogen has an unusually low chemical shift (d 3.40), suggesting that this hydrogen is close to the oxygen of the furan ring.3][34][35][36] The analysis of NOESY correlation map (acetone-d 6 ) allowed us to determine the relative configuration of C-11.Clear NOESY correlation was observed between the methinic proton H-7 (a-oriented) and the methylene hydrogens H-13a and H-13b attached to C-13 (Supplementary Information).Based on this observation, we can suggest a β-orientation for the hydroxyl group at C-11 (C−OH) and an a-orientation for the bond between the carbons C-11 and C-13 (Figure 1).
The 1 H and 13 C NMR data (Table 1) of compound 1 resembled those of sesquiterpene lactone 11,13-epoxycalaxin previously isolated from C. pilosa, 18 clearly indicating that both compounds present the same skeletal arrangement.These two sesquiterpene lactones differ  4).The compound 1 has been identified in the crude extract from C. pinnatifida by ultra-high performance liquid chromatography electrospray ionization mass spectrometry (UHPLC-ESI-MS), indicating that this compound isn't a product of ring opening of an epoxide from natural sesquiterpene lactone 11,13-epoxy-calaxin.
Compounds 3-5, 6, 8 and the mixture of 6 + 7 + 8 displayed weak or no percentage of inhibition against intracellular amastigotes of both T. cruzi and L. amazonensis, whereas the compound 1 exhibited a strong leishmanicidal and trypanocidal effect, inhibiting the intracellular parasite growth in 81.81 and 94.30%, respectively.Furthermore, this compound displayed IC 50 values of 5.27 ± 0.19 and 8.30 ± 0.57 μM for T. cruzi and L. amazonensis, respectively, showing a promising trypanocidal and leishmanicidal activity.
Regarding the cytotoxic potential on THP-1 cells, the sesquiterpene lactone 11,13-dihydroxy-calaxin presented a lower selectivity for the parasite cells (CC 50 < 15.60), resulting in low selectivity index (SI value not determined).Cytotoxicity (CC 50 ) and the selectivity index (SI) has not been calculated for the other compounds because all exhibited a weak leishmanicidal and trypanocidal effect (%growth inhibition < 50%).Cytotoxicity is an essential parameter for the initial stages of the development de new drugs, once that it determines the in vitro selectivity for the parasites and whether the bioactive compounds cause toxic effects on mammalian host cells.Thus, the cytotoxicity tests with natural products, mainly plant species, are an important strategy because natural products constitute a promising source of new bioactive compounds, revealing chemical scaffolds important for the discovery and development of new and selective antiprotozoal. 42,43

Conclusions
The phytochemical investigation from C. pinnatifida leaves led to the isolation and characterization of a new furanoheliangolide sesquiterpene lactone in addition to seven known compounds.It is important to note that all the isolated compounds are reported here for the first time for the C. pinnatifida species.Moreover, the antiprotozoal assays shown that the furanoheliangolide 11,13-dihydroxy-calaxin presented a remarkable trypanocidal and leishmanicidal effect against both T. cruzi and L. amazonensis amastigotes.

Figure 1 .
Figure 1.Chemical structures of the isolated compounds from C. pinnatifida.
1379 [M + H] + (calcd.for C 19 H 23 O 8 : 379.1393), and the negative mode HRESIMS spectrum revealed a molecular ion peak at m/z 377.1234 [M -H] -(calcd.for C 19 H 21 O 8 : 377.1236), both compatible with molecular formula C 19 H 22 O 8 , corresponding to nine degrees of unsaturation.The ESI-MS/MS spectral data of compound 1 revealed a base ion peak with m/z 291 (C 15 H 15 O 6 ) formed by the loss of methacrylate group [M -H -(CH 2 C(CH 3 )CO 2 )] -.Moreover, there were also produced m/z 247 (C 14 H 15 O 4 ) and m/z 229 (C 14 H 13 O 3