Bufadienolides from the Skin Secretions of the Neotropical Toad Rhinella alata (Anura: Bufonidae): Antiprotozoal Activity against Trypanosoma cruzi

Toads in the family Bufonidae contain bufadienolides in their venom, which are characterized by their chemical diversity and high pharmacological potential. American trypanosomiasis is a neglected disease that affects an estimated 8 million people in tropical and subtropical countries. In this research, we investigated the chemical composition and antitrypanosomal activity of toad venom from Rhinella alata collected in Panama. Structural determination using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy led to the identification of 10 bufadienolides. Compounds identified include the following: 16β-hydroxy-desacetyl-bufotalin-3-adipoyl-arginine ester (1), bufotalin (2), 16β-hydroxy-desacetyl-bufotalin-3-pimeloyl-arginine ester (3), bufotalin-3-pimeloyl-arginine ester (4), 16β-hydroxy-desacetyl-bufotalin-3-suberoyl-arginine ester (5), bufotalin-3-suberoyl-arginine ester (6), cinobufagin-3-adipoyl-arginine ester (7), cinobufagin-3-pimeloyl-arginine ester (8), cinobufagin-3-suberoyl-arginine ester (9), and cinobufagin (10). Among these, three new natural products, 1, 3, and 5, are described, and compounds 1–10 are reported for the first time in R. alata. The antitrypanosomal activity assessed in this study revealed that the presence of an arginyl-diacid attached to C-3, and a hydroxyl group at C-14 in the structure of bufadienolides that is important for their biological activity. Bufadienolides showed cytotoxic activity against epithelial kidney Vero cells; however, bufagins (2 and 10) displayed low mammalian cytotoxicity. Compounds 2 and 10 showed activity against the cancer cell lines MCF-7, NCI-H460, and SF-268.


Introduction
Compounds present in the venom of toads have shown potential as therapeutic agents for the treatment of human diseases such as cancer and cardiovascular diseases [1]. Historically, the crude venom has been used as ethnopharmacological treatment. For instance, in South America, indigenous tribes use the skin secretions from Rhinella marina and Rhinella jimi to prepare a remedy for cancer and inflammation [2,3]. The Chinese drug Chan Su, which is prepared using the skin secretion of toads such as Bufo gargarizans and Duttaphrynus melanostictus, is a millenary medicine used to treat chronic hepatitis B, heart failure, pain, and cancer [4].

Isolation and Structural Elucidation
The specimens of R. alata were collected at Parque Nacional Soberanía, Panama. Venom production was induced using electrical stimulation and collected in vials containing CH 3 OH. The methanolic extract of R. alata was subjected to activity-guided fractionation using solid phase extraction, followed by reversed phase HPLC purification, yielding compounds 1-10.
Compound 3 presented an optical activity ([α] 20 D ) of +47. The molecular formula of compound 3 was calculated as C 37 H 56 N 4 O 9 based on HR-ESI-QTOFMS spectra, which showed the molecular ion at m/z 701.4081 [M + H] + . Comparisons of the nmR data of compounds 1 and 3 (Table 1) indicated that compound 3 has the same steroidal core (Figures S10-S14 and Table S2). A difference of 14 Daltons in the molecular mass of compounds 1 and 3 suggested an additional methylene in compound 3, which may be located in the arginine/dicarboxylic acid moiety ( Figure S15). The MS/MS spectrum of compound 3 showed fragments at m/z 683.3954, 666.3944, 317.1829, and 175.1204, which are in agreement with the losses of two H 2 O molecules, pimeloyl-arginine, and the amino acid residue arginine, respectively ( Figure S16). Assignments of the structure of compound 3 were confirmed by 1D and 2D nmR data (Table 1, Figure 1) and, therefore, it was identified as 16β-hydroxy-desacetyl-bufotalin-3-pimeloyl-arginine ester.          Compound 5 presented an optical activity ([α] 20 D ) of +51. The molecular formula of compound 5 was determined as C 38 H 58 N 4 O 9 based on the molecular ion peak observed at m/z 715.4228 [M + H] + obtained by HR-ESI-QTOFMS. As compounds 1 and 3, the aglycone of compound 5 was assigned as 16β-hydroxy-desacetyl-bufotalin by comparing of the 1D and 2D nmR data with those of compounds 1 and 3 (Table 1, Figure 1). The molecular mass of compound 5 had 14 Daltons more than compound 3, indicating the presence of an additional methylene in the arginine/dicarboxylic acid moiety. The MS/MS spectrum of compound 5 showed peaks corresponding to the losses of water at m/z 697.4086 and 679.3975, suberoyl-arginine at m/z 331.1945, and arginine at m/z 175.1152. The HMBC spectrum of compound 5 showed correlations between H-3 (δ H 5.08) and C-1 (δ C 175.1), as well as for H-1" (δ H 4.42) to C-8 (δ C 175.1), indicating the linkages between the steroidal core to the dicarboxylic acid moiety, and this latter to the arginine residue ( Figure 1). Therefore, compound 5 was identified as 16β-hydroxy-desacetyl-bufotalin-3-suberoylarginine ester. For 1D-NMR, 2D-NMR and mass spectra of compound 5 see Figures S17-S24  and Table S3. NMR spectroscopy and MS analysis led to the chemical characterization and identification of three bufotoxins, 1, 3, and 5, which were not previously described, as far as we know, in the skin secretions of bufonids. Furthermore, five bufotoxins, 4 and 6-9, and two bufagins, 2 and 10, previously reported in bufonid toads were also found in the skin secretion of R. alata. nmR and MS spectroscopic data of known bufadienolides 2, 4, 6, 7, 8, 9 and 10 are included in the Supplementary Information. Bufotoxins 4 and 6-9 have been isolated previously from the skin venom of the Asiatic species of Bufo gargarizans and Bufo japonicus [9,10,23]. On the other hand, bufagins 2 and 10 have been found in species of the genera Atelopus, Bufo, Duttaphrynus, and Phrynoidis [24][25][26][27]. None of the compounds (1-10) identified in this study were found previously in the venom of species of the genus Rhinella. Moreover, marinobufagin and telocinobufagin, which are the most common constituents of the toad venom of South American Rhinella species, such as Rhinella crucifer, R. granulosa, R. icterica, R. jimi, R. margaritifera, R. major, R. marina, and R. schneideri [28], were not found in R. alata from Panama.

Antitrypanosomal Activity
The antitrypanosomal activity of bufadienolides 1-10 was tested in vitro against T. cruzi. The antiparasitic activity exhibited by each bufagin and bufotoxin assessed are presented in Table 2. Our results showed that substitution of the OH group at C-3 by the arginyl-diacid moiety increases trypanocidal efficacy, as observed when comparing compounds 2 and 10 with their bufotoxins analogues (4 and 6-9, respectively). The acetylation on the hydroxyl group at C-16 lightly increases the activity we observe in compounds 4 and 6 (IC 50 = 4.6 and 4.9 µM) compared with compounds 1, 3, and 5 (IC 50 = 6.9-8.7 µM). Moreover, the antitrypanosomal activity of bufadienolides with an OH group at C-14 was remarkably stronger than those with the epoxy group between C-14 and C-15. For example, bufotoxins 1 and 3-6 (4.6-8.7 µM) showed higher inhibition than bufotoxins 7-9 (14.7-19.0 µM). Similarly, the same biological property was observed when comparing bufagins 2 and 10, which exhibited IC 50 values of 19.6 µM and 29.0 µM, respectively. Tempone et al. (2008) [13] reported the antitrypanosomal activity of hellebrigenin against T. cruzi with an IC 50 value of 91.7 µg/mL against trypomastigotes. In the Tempone study, they also tested the activity of telocinobufagin; however, this compound did not present activity (IC 50 > 200 µg/mL). Based on these results, we suggest that the presence of the arginyl-diacid ester at C-3 and of an OH at C-14 are essential structural components of the bufadienolides required for the effective biochemical interactions with biological targets present in the parasites, which led to the antitrypanosomal activity observed.
Bufadienolides are known to inhibit the (Na + -K + ) ATPase pump through binding to the α-subunit. The inhibition provokes intracellular sodium concentration increasing, leading to depolarization of the cell [11]. In trypanosomes, however, there is no report on identification of this protein. Moreover, ionic steady-state maintenance is carried out by K + channels, Na + efflux pumps, and H + -ATPases [28]. Remarkably, a gene encoding for (Na + -K + ) ATPase from T. cruzi was cloned and characterized. After biochemical evaluations, it was revealed that enzymatic activity was not inhibited by the cardenolide ouabain [29]. These findings may suggest that bufadienolides inhibit the growth of T. cruzi parasites by a mechanism other than the blockage of the (Na + -K + ) ATPase. Parasitic diseases caused by protozoa also affect amphibians [30]. Species from different families of anurans, including Bufonidae, are the host of trypanosomes and frequently found to be parasitized by more than one species. The pathogenicity for amphibian trypanosomiasis has been described as anemia, food refusal, listlessness, and localized hemorrhages with swollen lymph glands. Bufonids biosynthesize bufadienolides, as shown by studies using cholesterol marked isotopically [31]. These compounds have been isolated from toads venom as well as from bile, eggs, ovary, and plasma [8,32,33]. Because they inhibit (Na + -K + ) ATPase pump, bufadienolides are considered as possible regulators of the ionic equilibrium in frogs and toads [24]. However, a possible role as endogenous antiparasitic substances should not be overlooked.

Mammalian Cytotoxicity
The cytotoxic activity of the bufadienolides 1-10 was evaluated in vitro against epithelial kidney monkey Vero cells (ATCC). The cytotoxicity exhibited by each bufadienolide is presented in Table 3. Bufadienolides conjugated at C-3 with arginil-dicarboxilic acid groups display higher cytotoxicity (IC 50 0.17-3.60 µM) than bufagins (IC 50 46-54 µM). In this sense, to decrease the cytotoxic effect, the microbial transformation of bufadienolides has allowed them to produce no cytotoxic analogues [34].

Anticancer Assay
The anticancer activity of compounds 2 and 10 was determined by absorbance after treatment of remnant living cells in three human cell lines. Their histopathological features are presented in Table 4. Both bufadienolides (2 and 10) exhibited cytotoxic activity against the tested three cancer lines with marked potency against MCF-7 cells, with compound 10 (IC 50 = 9 nm) being even more potent than the positive control. In Table 4 the data for the anticancer evaluation are displayed. The bufadienolides identified in this research have shown anticancer activity in vitro. Bufotalin-3-pimeoyl-arginyl ester (4) induced growth inhibition against liver cancer cells at 7.24 µM [9]. Cinobufagin (10) has shown inhibitory activity for lung, nasopharyngeal, and hepatic carcinomas [10,35,36]. Bufotalin (2) has presented activity against cervical and liver cancer cell lines [9,36]. The bufagins 2 and 10 induce cellular death by apoptosis [37,38]. In our study, compounds 2 and 10 inhibited the growth of MCF-7, NCI-H460, and SF-268 human cancer cell lines. Besides anticancer activity, both compounds demonstrated potency, especially for MCF-7 cancer cells.

General Experimental Procedures
Ultraviolet spectra were registered using a UV-2401 PC recording spectrophotometer (Shimadzu, Columbia, MD, USA). Infrared spectra were measured using a Platinum ATR Alpha instrument (Bruker, Billerica, MA, USA). Optical activities were determined on a digital P-2000 polarimeter (JASCO, Easton, MD, USA) at room temperature. Nuclear magnetic resonance (NMR) experiments were carried out at 25 • C on an Eclipse+ 400 Fourier transform spectrometer with field strength of 9.38 T and equipped with a 5 mm TH tunable probe (JEOL, Peabody, MA, USA). Chemical shifts (δ) were expressed in parts per million (ppm). Carbon multiplicities were determined by DEPT-90 and DEPT-135 experiments. COSY, HMBC, HSQC, and NOESY correlations were recorded employing pulse field gradients. nmR spectra were referenced based on the residual signal of CD 3 OD (δ H 3.31 and δ C 49.0) or CDCl 3 (δ H 7.26 and δ C 77.0) depending on the solvent employed to dissolve the sample. For HR-ESI-QTOF analysis, compounds were diluted in CH 3 OH and directly infused into a micrOTOF-QIII™ spectrometer (Bruker Daltonics, Billerica, MA, USA). Auto MS spectra were acquired in positive electrospray ionization mode in the range of 50 to 2500 mass to charge ratio (m/z) and capillary voltage was set at 4500 V. Nitrogen was used as the nebulizer gas at 2.

Collection of Animals and Venom Extraction
The specimens were collected at Parque Nacional Soberanía, Panama (9.134994 • N 79.722597 • W; and 9.075650 • N 79.658933 • O) and identified in situ by Dr. Roberto Ibáñez. Induction of the production of fresh toad venom from the parotoid glands was performed by stimulation of the animals using 3 to 5 V at 60 Hz for 30 s in the dorsal region using a homemade transcutaneous amphibian stimulator [39]. The extracted secretion was placed in borosilicate vials containing CH 3 OH and stored at −20 • C until analysis. Specimens of R. alata toads were returned alive to the same collection site. The collection of the toads was made under the authorization of the ministerio de Ambiente of Panama (permits SE/AQ-2-14 and SE/A-32-15).

Isolation of Bufadienolides
The methanolic soluble extract of R. alata was loaded onto Supelclean™ LC-18 cartridges (Supelco, Bellefonte, PA, USA) and fractionated by employing a stepwise gradient of 20%, 40%, 60%, 80%, and 100% of CH 3 OH in water. Methanol was removed by rotary evaporation under vacuum; and the remaining water was removed by lyophilization. Each fraction was diluted in CH 3 OH and the compounds present in the fractions were separated by liquid chromatography using reverse-phase high-performance liquid chromatography (RP-HPLC) on an Agilent 1100 HPLC equipped with a Diode Array detector 1200 series (Agilent, Santa Clara, CA, USA). A semi-preparative Synergi Hydro C18 column (250 × 10 mm, 4 µM, 80 Å) (Phenomenex ® , Torrance, CA, USA) was used. Elution was performed at 300 nm at a flow rate of 1 mL/min. The mobile phase consisted of trifluoroacetic acid (TFA) at 0.1% in water (Solvent A) and a 1/1 mixture of CH 3 OH/MeCN acidified with TFA at 0.1% (Solvent B). HPLC purification of the 60% CH 3 OH -SPE fraction allowed for the isolation of compounds 1 (12 mg, R t 61.0 min) and 2 (7.1 mg, R t 68.0 min). The chromatography of the 80% CH 3 OH fraction led to the purification of compounds 3

Antiprotozoal Activity against T. cruzi
The evaluation of the antiprotozoal activity against trypanosomes was carried out using the recombinant strain Tulahuen clone C4 of T. cruzi trypomastigotes (ATCC, Manassas, VA, USA). This strain expresses the β-galactosidase enzyme used as a reporter to assess the viability of the trypanosomatid [41]. The parasites were maintained at 37 • C under a 5% CO 2 atmosphere in RPMI-1640 culture medium with L-glutamine, 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer, NaHCO 3 , 10% FBS, and 0.05% gentamicin (50 mg/mL) as supplements. Vero cells were cultured for 24 h, and one day prior to the experiment, the cells were infected with trypomastigotes. After additional 24 h of infection, bufadienolides were dissolved in DMSO and tested at four different concentrations by duplicate for five days. Benznidazole was used as the positive control. To determine antitrypanosomal activity, 25 µL of chlorophenol-red-β-D-galactopyranoside (Roche Applied Science) (900 µM) were added to each well and allowed to react with the β-galactosidase of the remaining living parasites for 4.5 h. Absorbance was calculated at 570 nm using a microtiter plate reader (Sinergy HT, BioTek Instruments Inc, Winooski, VT, USA).

Mammalian Cytotoxicity
For cytotoxicity assays, Vero cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated in 96-well plates at 37 • C under a 5% CO 2 atmosphere, using RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) sterilized with 0.05% gentamicin (50 mg/mL) and supplemented with 10% FBS (fetal bovine serum; Gibco, Invitrogen, Carlsbad, CA, USA). Vero cells were allowed to adhere for 24 h prior to incubation for five days with the samples. During the treatment, DMSO was used as negative control, and samples were assayed at four different concentrations. After incubation, MTT (3-(4,5-di-methyl-thiazol-2-yl)-2,5-di-phenyl-tetra-zolium bromide) was added to each well, and absorbance was determined 4 h later at 570 nm using a color plate reader. Cytotoxicity was evaluated colorimetrically by calculating the ability of the remnant living Vero cells to reduce the pale yellow MTT into the black-purple formazan product, as described earlier [42]. All bioassays were performed in duplicate and the IC 50 values were determined employing the Data Analysis complement Wizard of Excel 2000 (Microsoft, Seattle, WA, USA).

Anticancer Assay
MCF-7, NCI-H460, and SF-268 cancer cells lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultivated in 96-well plates at 37 • C under a 5% CO 2 atmosphere using RPMI-1640 medium (Sigma-Aldrich, USA) sterilized with 0.05% gentamicin (50 mg/mL), supplemented with 10% FBS (fetal bovine serum; Gibco, Invitrogen, Carlsbad, CA, USA), and allowed to adhere for 24 h [43]. After incubation, cancer cells were treated with bufadienolides dissolved in DMSO at five different concentrations and analyzed by duplicate for 48 h. RPMI without cells was used as color control and doxorubicin was used as inhibition control. After treatment, cells were fixed with 25% tricholoroacetic acid (TCA) for 60 min, air dried, and stained with sulforhodamine-B (SRB). Washes with acetic acid at 1% were carried out to eliminate excess of SRB. Tris was added to dissolve the bound SRB. The plate was shaken and absorbance was read at 515 nm using a color plate reader.

Statistical Analysis
All antitrypanosomal and anticancer assays represent independent analysis. The data are presented as the inhibitory concentration (IC 50 ) ± standard error (SE). The statistical analysis for the IC 50 values was carried out employing the Data Analysis complement Wizard of Excel 2000 by adjusting the dose-response curve to a sigmoidal model (Microsoft, Seattle, WA, USA).

Conclusions
The chemical analysis of the skin secretions of the neotropical toad R. alata from Panama led to the isolation of 10 metabolites, of which 3 bufadienolides, 16β-hydroxydesacetyl-bufotalin-3-adipoyl-arginine ester, 16β-hydroxy-desacetyl-bufotalin-3-pimeloylarginine ester, and 16β-hydroxy-desacetyl-bufotalin-3-suberoyl-arginine ester, are reported for the first time not only in the genus Rhinella, but also among the Bufonidae family. The rest of the compounds identified in this study were found for the first time in the genus Rhinella. Most of the metabolites isolated belong to the bufotoxin class of bufadienolides. The antitrypanosomal activity assessed in this study revealed that the presence of an arginyl-diacid at C-3 and an OH group at C-14 in the structure of bufadienolides, which are important for the bioactivity observed. Bufadienolides show marked antitrypanosomal activity with significant cytotoxicity against normal cells. Additionally, bufotalin and cinobufagin exhibited anticancer activity against MCF-7, NCI-H460, and SF-268 cell lines.