Toxicarioside M, a new cytotoxic 10β-hydroxy-19-nor-cardenolide from Antiaris toxicaria

Fitoterapia 83 (2012) 660–664

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Toxicarioside M, a new cytotoxic 10β-hydroxy-19-nor-cardenolide from Antiaris toxicaria Claire Levrier a, Bernard Kiremire b, Françoise Guéritte a, Marc Litaudon a,⁎ a b

Centre de Recherche de Gif, Institut de Chimie des Substances Naturelles (ICSN), CNRS, LabEx LERMIT, 1 Avenue de la Terrasse, 91198 Gif-Sur-Yvette cedex, France Chemistry Department, Makerere University, Box 7062, Kampala, Uganda

a r t i c l e

i n f o

Article history: Received 22 November 2011 Accepted in revised form 3 February 2012 Available online 12 February 2012 Keywords: Antiaris toxicaria Cardenolide 10β-hydroxy-19-nor-cardenolide Toxicarioside M Cytotoxicity

a b s t r a c t A new 10β-hydroxy-19-nor-cardenolide, named toxicarioside M (1), was isolated from the trunk bark of Antiaris toxicaria (Pers.) Lesch (Moraceae), along with six known cardenolides (convallatoxin (2), convallatoxol (3), convalloside (4), 3-O-ß-D-xylopyranosylstrophanthidin (5), glucostrophanthidin (6) and strophanthidin (7)). Their structures were elucidated on the basis of HR-MS n analysis, spectroscopic methods (IR, UV, 1D and 2D NMR) and by comparison with data reported in the literature. The cardenolides were evaluated for their cytotoxic activity against KB, HCT-116, SF-268, MCF-7, HL-60, PC-3 and MRC-5 cell lines. © 2012 Elsevier B.V. All rights reserved.

1. Introduction With the objective to discover new bioactive compounds from the Ugandese flora, Antiaris toxicaria (Pers.) Lesch, of the Moraceae family, was selected for a phytochemical study following its potency against the KB cancer cell line. This species is extremely widespread in tropical regions from the West to East Africa and Madagascar, tropical Asia, and the western Pacific including Fiji and Tonga islands [1]. The bark yields a latex which is one of the principle components of most dart and arrow poisons in South-East Asia. In Africa, the latex of A. toxicaria (Moraceae) is applied to cuts, wounds and skin complaints such eczema and leprosy, and is taken internally as a purgative [1]. This species is known to produce mainly prenylphenols [2,3] and cardiac glycosides [4,5]. While cardenolides are more frequently associated with cardiovascular activity, it is only recently that several studies have demonstrated the effectiveness of certain members of this class of compounds for their anticancer

⁎ Corresponding author at: 1 Avenue de la Terrasse, 91198 Gif-Sur-Yvette, France. Tel.: + 33 1 69 82 30 85; fax: + 33 1 69 07 72 47. E-mail address: [email protected] (M. Litaudon). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2012.02.001

properties [6,7], and as evidenced by the recent patents, taken as potent cancer therapeutic agents [8,9]. Cardenolides bind and inhibit the membrane Na +/K + ATPase. This protein, which is overexpressed in some cancer cells, is implicated in the development and progression of different tumours [6]. In this context and taking into account that the cytotoxic activity of convallatoxol (3) and convalloside (4) has never been reported so far, we have completed the chemical investigation of A. toxicaria bark to assess the cytotoxic properties of its main constituents. In this report, we describe the isolation and characterisation of seven cardenolides, among which toxicarioside M (1) is new (Fig. 1), and evaluated their cytotoxicity activity against 6 cancer and one normal cell lines. 2. Experimental 2.1. General Optical rotations were measured at 25 °C on an Anton Paar MCP 300/500 polarimeter. IR spectra were obtained on a Perkin Elmer Spectrum BX FT-IR system. The NMR spectra of toxicarioside M (1) were recorded on a Bruker Avance

C. Levrier et al. / Fitoterapia 83 (2012) 660–664

were used for flash chromatography using a CombiFlash Companion apparatus (Serlabo, France).

O O

R1 OH R2

661

OH

Toxicarioside M (1): R1 = OH, R2 = Rha Convallatoxin (2): R1 = CHO, R2 = Rha Convallatoxol (3): R1 = CH2OH, R2 = Rha Convalloside (4): R1 = CHO, R2 = Rha (4 1) Glc Xylostrophanthidin (5): R1 = CHO, R2 = Xyl Glucostrophanthidin (6): R1 = CHO, R2 = Glc Strophanthidin (7): R1 = CHO, R2 = OH Fig. 1. Cardenolides isolated from Antiaris toxicaria trunk bark. Rha = α- L-rhamnopyranosyl; Glc = β-D-glucopyranosyl; Xyl = ß-D-xylopyranosyl.

600 MHz instrument, using DMF-d7 at 223 K and DMSO-d6 at 298 K. The NMR spectra of the other compounds were recorded on a Bruker Avance 500 MHz instrument (Bruker; Switzerland), using DMSO-d6 and pyridin-d5 as solvents. High resolution MS data were obtained on a LCT Premier XE (Waters) ESI-ToF mass spectrometer, using a Waters Acquity UPLC. LC–electrospray high-resolution tandem mass spectrometry experiments were realised using a HPLC system (Ultimate 3000, Dionex) coupled to a hybrid linear trap/orbitrap mass spectrometer (LTQ-orbitrap, ThermoFisher, Germany) equipped with an electrospray source. Analyses were performed in negative ionisation mode. Experimental parameters were as follows: the spray tension was set at 3.1 kV, capillary temperature at 300 °C, capillary voltage at −25 V, tube lens voltage at −65 V, and finally gate lens voltage at 70 V. Resolution was fixed at 60,000 for MS analysis and 30,000 for MS/MS analysis. Exact mass measurements were obtained with a precision better than 2 ppm and 5 ppm for MS and MS/MS, respectively. Separation was performed with a C18 column Sunfire (5 μm; 2.1× 150 mm) (Waters) using ACN-H2O gradient (from 5/95 to 100/0) under 250 μl/min flow rate, 95 bar and UV detection fixed at 210 nm. Kromasil analytical, semipreparative and preparative C18 columns (5 μm; 250 ×4.6 mm; 250 × 10 mm and 250 × 21.2 mm; Thermo), and Symmetry Shield preparative C18 column (5 μm; 19× 150 mm, Waters), were used for preparative HPLC separations using a Waters autopurification system equipped with a sample manager (Waters 2767), a column fluidics organizer, a binary pump (Waters 2525), a UV–vis diode array detector (190–600 nm, Waters 2996), and a PL-ELS 1000 ELSD Polymer Laboratory detector. Silica gel 60 (6–35 μm) and analytical TLC plates (Si gel 60 F 254) were purchased from SDS (France). Kedde reagent (3,5-dinitrobenzoic acid + NaOH) was used to reveal cardenolides. Prepacked C18 Versapack (40 × 150 mm) cartridges

2.2. Plant material Trunk barks of A. toxicaria were collected in Kibale Forest National Park (Uganda) in August 2007, and identified by John Kasenene. A voucher specimen (UFC-0081) is deposited at the Herbarium of Makerere University, Uganda. 2.3. Extraction and isolation The powder air-dried bark (824 g) of A. toxicaria was extracted successively with EtOAc, MeOH and H2O–MeOH (50:50) (each 3 × 1 L, 60 min, room temperature). The crude extracts were concentrated in vacuo at 40 °C to yield 2.7 g, 4.7 g and 7.3 g of residue, respectively. Given that cardenolides were detected in the three extracts (positive to Kedde reagent), they were combined to afford one crude extract (14.5 g). A part of this extract (12.5 g) was subjected to flash chromatography using a gradient of H2O–MeOH (90:10 to 0:100) at 30 mL/min to afford 9 fractions according to their TLC profiles. Purification of fraction 3 (819 mg, H2O– MeOH 70:30), using a Symmetry Shield preparative C18 column (5 μm; 19 × 150 mm), H2O–ACN 85:15 at 17 mL/min yield to glucostrophanthidin (6) (22 mg, tR: 20 min). Fraction 5 (1.4 g, H2O–MeOH 55:45) was subjected to a silica gel column chromatography to afford strophanthidin (7) (17 mg), convallatoxin (2) (597 mg) and fractions 5A–I. Convalloside (4) (40 mg, tR: 6 min), toxicarioside M (1) (7 mg, tR: 10 min), convallatoxol (3) (20 mg, tR: 11 min) and 3-O-ß-D-xylopyranosylstrophanthidin (5) (14 mg, tR: 13 min) were purified from fraction 5 F with a Kromasil preparative C18 column (5 μm; 250 × 21.2 mm; Thermo), using H2O–ACN 77:23 + 0.1% formic acid at 21 mL/min. 2.3.1. Toxicarioside M (1) White amorphous powder; [α] 25D = − 10° (c = 0.1, MeOH); ESI-HRMS m/z 537.2714 [M-H] − (Calcd for C28H41O10 537.2699); UV (MeOH) λmax (logε) 210 (2.41) nm; IR νmax cm − 1: 3220, 1700, 1635; 1H and 13C NMR: Table 1. 2.4. Cytotoxicity activity assay KB (nasopharyngeal epidermoid carcinoma) and SF-268 (glioblastoma) were obtained from NCI; HCT-116 (colon adenocarcinoma) and HL-60 (acute promyelocytic leukaemia) were purchased from ATCC; PC-3 (prostate adenocarcinoma) and MRC-5 (human diploid embryonic lung cell) were purchased from ECACC, and MCF-7 (breast adenocarcinoma) was kindly provided by Matthias Kassack (University of Bonn). The chemicals were dissolved in DMSO and were added for 72 h to plate containing cells in a fixed volume of DMSO (1% final concentration). Controls received an equal volume of DMSO. The number of viable cells measured at 490 nm with the MTS reagent (Promega, Madison, WI) and IC50 was calculated as the concentration of compound eliciting a 50% inhibition of cell proliferation, according to a published procedure [10]. Docetaxel (in-house product, purity > 99%) was used as a reference compound.

662 Table 1 1 H (600 MHz) and

C. Levrier et al. / Fitoterapia 83 (2012) 660–664

13

C (125 MHz) NMR data in DMSO-d6 for compound 1.

Position

δH (J = in Hz)

δC

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 20 21 22 23 1′ 2′ 3′ 4′ 5′ 6′ OH-5⁎ OH-10⁎ OH-14⁎

1.76/1.56 1.53/1.63 3.93 1.62/1.94

28.8 27.7 72.7 35.0 73.7 34.6 23.2 39.6 39.0 73.2 20.6 38.9 49.2 83.6 32.0 26.4 50.0 15.7 174.0 73.3 116.2 176.6 99.0 70.8 70.8 72.0 69.1 17.9

1.23/1.69 0.94/1.81 1.75 m 1.36 m 1.25/1.37 1.44/1.35

1.54/2.0 1.77/1.99 2.72 m 0.76 s 4.88 d (18.4)/4.96 d (18.4) 5.90 s 4.70 s 3.58 s 3.41 dd (9.2. 3.0) 3.20 t (9.2) 3.43 dd (9.2. 6.4) 1.11 d (5.9) 4.22 4.05 4.62

⁎ Chemical shifts only observed in DMF-d7 at 223 K.

2.5. Acid hydrolysis Convallatoxin (20 mg) was dissolved in methanol (2 mL) and refluxed with HCl 1 N for 1 h. The reaction mixture was evaporated to dryness under vacuum and then partitioned between EtOAc and H2O. The H2O layer was concentrated to yield α-L-rhamnose. The α-L-rhamnose was identified by comparison with authentic sample using Supercritical Fluid Chromatography (SFC). The SFC analysis was performed on a Thar Waters SFC Investigator II System using a Waters 2998 photodiode array detector and a Princeton Cyano Column (5 μM; 256 × 4.6 mm) equipped with a Waters Acquity ELSD 2424 detector, with the following conditions: CO2–MeOH 95:5, 4 mL/min, 150 bar. 3. Results and discussion 3.1. Chemistry The powdered bark of A. toxicaria (824 g) was extracted successively with EtOAc, MeOH and H2O/MeOH (50:50). These extracts were combined to give a dry crude extract (14.5 g) after removal of the solvents. The combined extracts exhibited a significant cytotoxic activity (70% at 1 μg/mL) on KB cells. We then performed a chemically-guided fractionation on the crude extract, using Kedde reagent, to afford toxicarioside M (1), convallatoxin (2), convallatoxol (3), convalloside (4), xylostrophanthidin (5), glucostrophanthidin (6) and strophanthidin (7) (Fig. 1).

Compound (1) was isolated as a white amorphous powder. Its structure was established unambiguously upon analysis of LC-HRMSn, 1H and 13C NMR, COSY, ROESY, HMQC, and HMBC data. Its IR spectrum exhibited absorption bands for hydroxyl (3220 cm − 1), α,β-unsaturated lactone ring (1700 cm − 1), and olefinic group (1625 cm − 1). The UV spectrum indicated the presence of an α,β-unsaturated lactone ring (λmax 210 nm). Its molecular formula was determined as C28H42O10 from the deprotonated ion [M-H] − at m/z 537.2714 (Calcd for C28H41O10 537.2699), of which 8 degrees of unsaturation could be deduced. An ESI-MS n analysis of compound 1 indicated the presence of a deoxysugar moiety and four hydroxy groups. In negative ionisation mode, the MS 2 product ion spectrum from the pseudomolecular ion [M-H] − of 1 showed a fragment ion at m/z 391.2122 (C22H31O6), corresponding to the loss of a deoxysugar moiety by cleavage of the osidic bond between the anomeric carbon and the oxygen. From the ion at m/z 391.2122, the MS 3 product ion spectrum showed peaks at m/z 373.2015 (C22H29O5), 355.1909(C22H27O4), 337.1804 (C22H25O3) and 319.1696 (C22H23O2), corresponding to the successive loss of 4 molecules of water [M-H − nH2O] −, thus indicating the presence of four hydroxy groups on the genin. The NMR data of 1 showed close similarities with those obtained for convallatoxin (2). It can be deduced that compound 1 lacked an aldehyde group but possessed a hydroxy group instead. The analysis of 1H NMR data (Table 1) showed characteristic signals for α,β-unsaturated lactone ring at δH 5.90 (1H, brs, H-22), δH 4.88 (1H, d, 18.4 Hz, H-21a) and δH 4.96 (1H, d, 18.4 Hz, H-21b), an anomeric signal at δH 4.70 (1H, s, H-1′), together with resonances at δH 3.58 (1H, s, H-2′), δH 3.41 (1H, dd, 9.2 Hz, 3.0 Hz, H-3′), δH 3.20 (9.2 Hz, t, H-4′), δH 3.43 (1H, dd, 9.2 Hz, 6.4 Hz, H-5′) and δH 1.11 (3H, d, 5.9 Hz, H-6′), which suggested the presence of a α-Lrhamnopyranosyl moiety. The 13C NMR spectrum of compound 1 (Table 1) revealed the presence of 28 carbons, which were identified as two methyls, ten methylenes, ten methines and six quarternary carbons. In the HMBC spectrum (Fig. 2), key correlations between H-17 and C-13 (δC 49.2), C-14 (δC 83.6) and C-22 (δC 116.2), between the CH3-18 protons and C-13, C-14 and C-17, between H-2 and H-8 with C-10 (δC 73.2), and between H-3 and OH-10 with C-5 (δC 73.7), allowed to propose the planar structure of the genin. The attachment of the α-L-rhamnopyranose unit 3 at C-3 on the aglycone was established by the J correlation between the anomeric proton H-1′ and the oxymethine C-3 at δC 72.7. The relative stereochemistry of compound 1 was determined with ROESY experiment in DMF-d7 at 223 K. Key correlations (Fig. 3) between OH-10 and, H-8, H-3′ and H-5′ (in axial position), between H-9 and H-17, and between CH3-18 and, H-8 and OH-14 (in axial position) confirmed the stereochemistry of compound 1 predicted in Fig. 2. These data indicated that the A/B and C/D ring junctions are cis, with OH-5, OH-10, OH-14 and CH3-18 groups located on the β face. Finally, the ROESY correlation between H-3 with the anomeric proton confirmed the β-linkage of the αL -rhamnopyranose on the genine. Thus, compound 1, which was named toxicarioside M, was assigned the structure 10βhydroxy-19-norperiplogenin-3β-O-α-L-rhamnopyranose. To our knowledge, toxicarioside M (1) is the third example of 10β-hydroxy-19-nor-cardenolide discovered in nature [11,12].

C. Levrier et al. / Fitoterapia 83 (2012) 660–664

663

3.3. Cytotoxicity

Fig. 2. Key HMBC correlations for compound 1.

The formation of the OH-10 group could come from the autoxidation of the aldehyde group (C-19) into a carboxylic acid group followed by a spontaneous decarboxylation and an oxidation of the carbon at position 10. Convallatoxin (2), convallatoxol (3), convalloside (4), 3O-ß-D-xylopyranosylstrophanthidin (5), glucostrophanthidin (6) and strophanthidin (7) were also isolated from A. toxicaria trunk bark. These compounds were identified by comparison of their physical and spectroscopic data with those reported in the literature [13–19,22].

3.2. Acid hydrolysis Given the identical NMR data of the sugar moiety for convallatoxin and compound 1, and that convallatoxin was isolated in large amount, we verified the L-configuration of the rhamnose of convallatoxin (2). Its hydrolysis gave L-rhamnose and strophanthidin (7). Identification of L-rhamnose, was carried out by direct SFC analysis of the hydrolysate against a reference of L-rhamnose (Sigma).

Cardenolides 1–7 were subjected to a cytotoxic assay against KB, HCT-116, MCF-7, PC-3, SF-268, HL-60 and MRC-5. As shown in Table 2, most of the cardenolides (2–4 and 6, 7) exhibited potent cytotoxic activities. It is suggested from these results that the presence of a rhamnopyranose or a glucopyranose unit at position 3, together with an aldehyde function or at a lesser extent a primary alcohol at position 10, play an important role in the cytotoxic activity (toxicarioside M (1), xylostrophanthidin (5) and strophanthidin (7) were found less active). Although cardenolides 2–7 are oxidised at C-19, our results on MCF-7 are consistent with those of Rashan et al. [20], who showed that monoglycosidic cardenolides possessing the 3β,14βdihydroxy-5β-card-20(22)-enolide structure are the most effective cytotoxic compounds isolated from Nerium oleander and Streptocaulon tomentosum. In case of A. toxicaria, for the glycosylated compounds having an aldehyde group at C-10 (2, 4–6), convallatoxin (2) and glucostrophanthidin (6), which possess α-L-rhamnosyl and β-D-glucosyl residues respectively, exhibited the strongest cytotoxic activity with IC50s in the nM range, while xylostrophanthidin (5) and convalloside (4) having β-D-xylopyranosyl and α-L-rhamnopyranosyl-(4→1)β-D-glucopyranosyl respectively, are less cytotoxic. In addition, since IC50 values of convallatoxin (2) and strophanthidin (7) are much lower than those of convallatoxol (3), and 17α-Hperiplogenin [20], respectively (0.002, 0.66, 0.34 and 5.3 μM, respectively), it could be deduced that the aldehyde group play an important role in the cytotoxic activity. These results are consistent with those of Wang et al. [22], who showed that xylostrophanthidin (5) had stronger cytotoxic activities than 3-O-ß-D-xylopyranosylperiplogenin on BGC-823 (human gastric cancer cells) and Bel-7402 (human hepatoma cells) (IC50 0.016 and 0.13 µM for compound 5, and 0.23 and 0.48 µM for 3-O-ß-D-xylopyranosylperiplogenin, respectively). Previously, convallatoxin was found to be cytotoxic on HSG (human submandibular gland carcinoma, IC50 28 nM) [21], on BGC-823 (human gastric cancer cells, IC50 16 nM) [22], Bel7402 cells (human hepatoma cells, IC50 50 nM) [22], on NB12 (neuroblastoma stem cells, IC50 73 nM) [9] but not on HPLF (human periodontal ligament fibroblasts) [21]. Finally, in contrast with toxicarioside H [11], which exhibited strong cytotoxic activities (IC50s ranged from 8 to 70 nM on various cancer cell lines), the cytotoxic activity of toxicarioside M (1) is moderate (IC50s from 1.5 to 9 μM). Both toxicariosides H and M are monoglycosylated 19-nor-cardenolides but only toxicarioside H possess an additional β-hydroxy group at C-12.

4. Conclusion

Fig. 3. Key ROESY correlations for compound 1.

Phytochemical investigation of A. toxicaria led to the isolation of seven cardenolides, of which toxicarioside M (1) is a new 10β-hydroxy-19-nor-cardenolide. Compounds 2–4 and 6, 7 showed potent cytotoxic activity against KB, HCT-116, SF-268, MCF-7, HL-60, PC-3 and MRC-5 cell lines. Structureactivity relationships within this class of compounds have revealed that a potent cytotoxic activity is associated with the presence of a monoglycoside unit at C-3 and an aldehyde function at C-10 on the aglycone.

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Table 2 Cytotoxic activity of cardenolides 1–7 and Docetaxel as reference, against various cancer cell lines (IC50s in nM). Compound

Toxicarioside M (1) Convallatoxin (2) Convallatoxol (3) Convalloside (4) Xylostrophanthidin (5) Glucostrophanthidin (6) Strophanthidin (7) Docetaxel a

Cell linesa KB

HC-T116

MCF-7

PC-3

SF-268

HL-60

MRC-5

1540 0.52 105 65 840 17.5 190 0.16

3545 0.65 105 50 600 14.8 175 0.52

3920 2.20 345 225 2135 9.3 665 1.00

5930 2.10 185 290 1565 87.0 750 1.40

2220 0.40 65 45 750 119.0 220 0.59

8890 1.30 270 155 2210 108.0 415 0.46

2275 0.50 110 45 865 16.0 175 0.72

IC50s are mean values from two replicates (the variation is a maximum of 20%).

Acknowledgement The authors would like to thank John Kasenene (Ouganda) for collecting and identifying the plant material. They also acknowledge Marie-Thérèse Martin (ICSN) and JeanFrancois Gallard (ICSN) for running NMR experiments, Isabelle Schmitz-Afonso (ICSN), Olinda Gimello (ICSN) and Nicolas Elie (ICSN) for running MS experiments and Geneviève Aubert (ICSN) for the cytotoxicity experiments. This work was done within the framework of the “Collaborative phytochemical research using selected flora and fauna species in Uganda” (CNRS, MNHN, MU and UWA) program and the authors are grateful to the Uganda Wildlife Authority (UWA) and to the Uganda National Council for Science and Technology (UNCST) for authorization to conduct scientific research in Uganda.

References [1] Bosu PP, Krampah E. Antiaris toxicaria Lesch. In: Louppe D, OtengAmoako AA, Brink M, editors. Prota 7(1): Timbers/Bois d'œuvre 1. [CD-Rom]. Wageningen, Netherlands: PROTA; 2005. [2] Hano Y, Mitsui P, Nomura T. Seven prenylphenols, antiarones C, D, E, F, G, H and I from the root bark of Antiaris toxicaria Lesch. Heterocycles 1990;31:1315–23. [3] Hano Y, Mitsui P, Nomura T, Kawai T, Yoshida Y. Two new dihydrochalcone derivatives, antiarones J and K, from the root bark of Antiaris toxicaria. J Nat Prod 1991;54:1049–55. [4] Müuhlradt P, Weiss E, Reichstein T. Die cardenolide der samen von Antiaris toxicaria Lesch. 1. Mitteilung: isolierungen und identifizierungen. Helv Chim Acta 1964;47:2164–85. [5] Kiliani H. Über den milchsaft von Antiaris toxicaria. Berichte der deutschen chemischen gesellschaft. Helv Chim Acta 1910;43:3574–9. [6] Mijatovic T, Van Quaquebeke E, Delest B, Debeir O, Darro F, Kiss R. Cardiotonic steroids on the road to anti-cancer therapy. Biochim Biophys Acta 2007;1776:32–57.

[7] Newman RA, Yang P, Pawlus AD, Block KI. Cardiac glycosides as novel cancer therapeutic agents. Mol Interv 2008;8:36–49. [8] R. Valdes, K. Ihenetu, R. Fernandes-Botran, and H. Qazzaz, Treating cancer with cardiac glycosides, patent n°12/131,763, U.S. Patent 12/131,7632009. [9] L. M. Hansford, K. M. Smith, A. Datti, F. M. Miller, and D. R. Kaplan, Cancer stem cells and uses thereof, patent N°12/608,310, U.S. Patent 12/608,3102010. [10] Tempete C, Werner GH, Favre F, Rojas A, Langlois N. In vitro cytostatic activity of 9-demethoxyporothramycin B. Eur J Med Chem 1995;30: 647–50. [11] Dai H-F, Gan Y-J, Que D-M, Wu J, Wen Z-C, Mei W-L. A new cytotoxic 19-nor-cardenolide from the latex of Antiaris toxicaria. Molecules 2009;14:3694–9. [12] Roy MC, Chang F-R, Huang H-C, Chiang MY-N, Wu Y-C. Cytotoxic principles from the formosan milkweed, Asclepias curassavica. J Nat Prod 2005;68:1494–9. [13] Shi L-S, Liao Y-R, Su M-J, Lee A-S, Kuo P-C, Damu AG, et al. Cardiac glycosides from Antiaris toxicaria with potent cardiotonic activity. J Nat Prod 2010;73:1214–22. [14] Wehrli W, Schindler O, Reichstein T. Die glykoside des milchsaftes von Antiaris toxicaria Lesch. aus Malaya sowie von Antiaris africana Engl. aus Kenya. Isolierungen. Helv Chim Acta 1962;45:1183–205. [15] Ahmad VU, Basha A. Spectroscopic data of steroid glycoside: cardenolides and pregnanes1st ed. ; 2006. New York. [16] Hashem A, Khalique A. Corchoside C-A new bitter glycoside from seeds of Corchorus capsularis L. Bangladesh J Sci Ind Res 1978;13:127–32. [17] Makarevich IF. Cardenolide glycosides of Cheiranthus allioni VII. Chem Nat Compd 1972;8:191–5. [18] Kopp B, Krenn L, Kubelkaqa E, Kubelka W. Cardenolides from Adonis aestivalis. Phytochemistry 1992;31:3195–8. [19] Zhernoklev KV, Slyusarskaya TV, Yarmolenko GN. Cardenolides of Strophanthus kombe III. 17α-strophadogenin. Chem Nat Compd 1993;29: 642–6. [20] Rashan LJ, Franke K, Khine MM, Kelter G, Fiebig HH, Neumann J, et al. Characterization of the anticancer properties of monoglycosodic cardenolides isolated from Nerium oleander and Streptocaulon tomentosum. J Ethnopharmacol 2011;134:781–8. [21] Higano TH, Kuroda MK, Sakagami HS, Mimaki YM. Convallasaponin A, a new 5β-spirostanol triglycoside from the rhizomes of Convallaria majalis. Chem Pharm Bull 2007;55:337–9. [22] Wang T-M, Hojo T, Ran F-X, Wang R-F, Wang R-Q, Chen H-B, et al. Cardenolides from Saussurea stella with cytotoxicity toward cancer cells. J Nat Prod 2007;70:1429–33.