In vitro cytotoxic, antifungal, trypanocidal and leishmanicidal activities of acetogenins isolated from Annona cornifolia A. St. -Hil. (Annonaceae)

LUCIANA A.R.S. LIMA TÂNIA M.A. ALVES CARLOS L. ZANI POLICARPO A. SALES JÚNIOR ALVARO J. ROMANHA SUSANA JOHANN PATRÍCIA S. CISALPINO LÚCIA P.S. PIMENTA MARIA AMÉLIA D. BOAVENTURA About the authors

Abstracts

Annona cornifolia A. St. -Hil. is a small annual perennial tree found in the Brazilian savannah; their green fruit is popularly used in the treatment of ulcers. The acetogenins isolated from the seeds of Annona cornifolia previously showed to possess antioxidant activity. In continuation of our investigations on the biological activities of acetogenins, four binary mixtures and ten pure adjacent bis-tetrahydrofuran annonaceous acetogenins were evaluated: the cytotoxic (against three human tumor cell lines), antifungal (against Paracoccidioides brasiliensis), trypanocidal (against Trypanosoma cruzi) and leishmanicidal (against Leishmania amazonensis) activities. Acetogenins presented cytotoxic activity confirming their potential use in anti-cancer therapy. Regarding leishmanicidal and trypanocidal activities, an inhibition of 87% of L. amazonensis amastigotes and 100% of T. cruzi amastigotes and trypomastigotes was observed, when tested at the concentration of 20 µg mL–1. Moreover, six acetogenins showed more activity against all the three tested isolates of P. brasiliensis than trimethoprim-sulfamethoxazole, a drug used for treating paracoccidioidomycosis. Thus, acetogenins may be an alternative in treating a number of diseases that have a huge impact on millions of people worldwide. This paper reports for the first time the antifungal, leishmanicidal and trypanocidal activities for these acetogenins.

Annona cornifolia ; acetogenins; cytotoxic; antifungal; trypanocidal; leishmanicidal


Annona cornifolia A. St. -Hil. é uma pequena árvore anual perene encontrada no cerrado brasileiro; seu fruto verde é usado popularmente no tratamento de úlceras. As acetogeninas isoladas a partir das sementes de Annona cornifolia anteriormente apresentaram atividade antioxidante. Continuando as nossas investigações sobre as atividades biológicas das acetogeninas, quatro misturas binárias e dez acetogeninas bis-tetra-hidrofurânicas adjacentes puras foram avaliadas quanto às atividades: citotóxica (sobre três linhagens de células tumorais humanas), antifúngica (sobre Paracoccidioides brasiliensis), tripanocida (sobre Trypanosoma cruzi) e leishmanicida (sobre Leishmania amazonensis). As acetogeninas apresentaram atividade citotóxica, confirmando seu uso potencial na terapia anticâncer. Quanto às atividades leishmanicida e tripanocida, foi observada uma inibição de 87% das formas amastigotas de L. amazonensis e 100% de formas amastigotas e tripomastigotas de T. cruzi, quando testadas a uma concentração de 20 µg mL–1. Além disso, seis acetogeninas mostraram maior atividade sobre todas as três cepas testadas de P. brasiliensis que o trimetoprim-sulfametoxazol, um medicamento usado no tratamento da paracoccidioidomicose. Assim, as acetogeninas podem ser uma alternativa no tratamento de uma série de doenças que têm um enorme impacto em milhões de pessoas no mundo. Este trabalho relata pela primeira as atividades antifúngica, leishmanicida e tripanocida para estas acetogeninas.

Annona cornifolia ; acetogeninas; citotóxica; antifúngica; tripanocida; leishmanicida


INTRODUCTION

Several Annona species of the Annonaceae family produce edible fruits that are widely consumed in Brazil, such as Annona squamosa and Annona muricata. Annona cornifolia A. St. -Hil., with orange fruits, is a small annual perennial tree found in the Brazilian savannah; their green fruit is popularly used in the treatment of ulcers (Correa 1984Correa MP. 1984. Dicionário de plantas úteis do Brasil e de plantas exóticas cultivadas, Rio de Janeiro: Imprensa Nacional 1: 194.). Annonaceous acetogenins, isolated exclusively from the Annonaceae family until 2008 (Pettit et al. 2008Pettit GR, Mukku VJRV, Cragg G, Herald DL, Knight JC, Herald CL and Chapuis JC. 2008. Antineoplastic agents. 558. Ampelocissus sp. Cancer cell growth inhibitory constituents. J Nat Prod 71: 130-133.), exhibit a broad range of biological properties (Yang et al. 2009Yang H, Zhang N, Li X, Chen J and Cai B. 2009. Structure-activity relationships of diverse annonaceous acetogenins against human tumor cells. Bioorg Med Chem Lett 19: 2199-2202.). Our group has already investigated acetogenins from the seeds of A. crassiflora(Santos et al. 1994Santos LP, Boaventura MAD and Oliveira AB. 1994. Crassiflorina, uma acetogenina tetra-hidrofurânica citotóxica de Annona crassiflora (araticum). Quím Nova 17: 387-391., 1996Santos LP, Boaventura MAD, Sun NJ, Cassady JM and Oliveira AB. 1996. Araticulin, a new bis-tetrahydrofuran polyketide from Annona crassiflora seeds. Phytochemistry 42: 705-707.) and A. cornifolia(Santos et al. 2006Santos LAR, Boaventura MAD and Pimenta LPS. 2006. Cornifolin, a new bis-tetrahydrofuran annonaceous acetogenin from Annona cornifolia. Biochem Syst Ecol 34: 78-82., 2007Santos LAR, Boaventura MAD and Pimenta LPS. 2007. Acetogeninas de anonáceas bioativas isoladas das sementes de Annona cornifolia A. St. -Hil. Rev Bras Plantas Med 9: 48-51., Lima et al. 2009Lima LARS, Pimenta LPS and Boaventura MAD. 2009. Two new adjacent bis-tetrahydrofuran annonaceous acetogenins from seeds of Annona cornifolia. Planta Med 75: 80-83., 2010Lima LARS, Pimenta LPS and Boaventura MAD. 2010. Acetogenins from Annona cornifolia and their antioxidant capacity. Food Chem 122: 1129-1138.), as well as from the leaves of Rollinia laurifolia (Pimenta et al. 2001Pimenta LPS, Nascimento FC, Assunção ACS, Oliveira AB and Boaventura MAD. 2001. Laurifolin, a novel acetogenin from Rollinia laurifolia leaves. Tetrahedron Lett 42: 8433-8434., 2003Pimenta LPS, Nascimento FC, Assunção ACS and Boaventura MAD. 2003. Acetogeninas de anonáceas isoladas de folhas de Rollinia laurifolia. Quím Nova 26: 319-322., 2005Pimenta LPS, Nascimento FC and Boaventura MAD. 2005. Acetogenins from the leaves of Rollinia laurifolia. Helv Chim Acta 88: 3225-3231.). There are no reports on the study of other parts of Annona cornifolia, including the phytochemical studies of the fruit. The fruits of several Annona species are an important source of proteins, carbohydrates and amino acids (Correa 1984Correa MP. 1984. Dicionário de plantas úteis do Brasil e de plantas exóticas cultivadas, Rio de Janeiro: Imprensa Nacional 1: 194.). Further research may shed light on the use of fruit of A. cornifolia in future dietary supplementation.

Leishmaniasis and trypanosomiasis, occurring in tropical and sub-tropical areas around the world, have huge medical, social and economic impact to millions of people (Castillo-Garit et al. 2012Castillo-Garit JA, Abad C, Rodrígues-Borges JE, Marrero-Ponce Y and Torrens F. 2012. A review of QSAR studies to discover new drug-like compounds against leishmaniasis and trypanosomiasis. Curr Top Med Chem 12: 852-865.). Paracoccidioidomycosis, a systemic disorder caused by the dimorphic fungus, Paracoccidioides brasiliensis, occurs in Latin American countries, resulting in more deaths than leishmaniasis in Brazil (Shikanai-Yasuda et al. 2006Shikanai-Yasuda MA, Telles Filho FQ, Mendes RP, Colombo AL and Moretti ML. 2006. Guideliness in paracoccidioidomycosis. Rev Soc Bras Med Trop 39: 297-310.). In the absence of drug therapy, paracoccidioidomycosis is usually fatal (Shikanai-Yasuda et al. 2006Shikanai-Yasuda MA, Telles Filho FQ, Mendes RP, Colombo AL and Moretti ML. 2006. Guideliness in paracoccidioidomycosis. Rev Soc Bras Med Trop 39: 297-310., Johann et al. 2010Johann S, Cisalpino PS, Watanabe GA, Cota BB, De Siqueira EP, Pizzolatti MG, Zani CL and De Resende MA. 2010. Antifungal activity of extracts of some plants used in the Brazilian traditional medicine against the pathogenic fungus Paracoccidioides brasiliensis. Pharm Biol 48: 388-396.). In the treatment of cancer, chemotherapeutic agents exhibit severe toxicity and can cause many undesirable side effects. Therefore, it is necessary to find new drugs, with natural products being good candidates. In fact, from 1983 to 1994, more than 60% of approved anticancer drugs were derived from natural products (Newman et al. 2003Newman DJ, Cragg GM and Snader KM. 2003. Natural products as sources of new drugs over the period 1981-2002. J Nat Prod 66: 1022-1037.).

In the present study, we evaluated the cytotoxic, antifungal, trypanocidal and leishmanicidal activities of fifteen adjacent bis-tetrahydrofuran (THF) acetogenins: ten pure (3-8 and 11-14) and four binary mixtures (1+2, 8+9, 8+10 and 8+15), isolated from the ethanol extracts of seeds from A. cornifolia A. St. -Hil. (Fig. 1).

Figure 1 -
Acetogenins isolated from Annona cornifolia. A) Acetogenins 1-10, B)Acetogenins 11-15.

MATERIALS AND METHODS

General

Etoposide, amphotericin B, sulforhodamine B, tris[hydroxymethyl]aminomethane buffer, trichloracetic acid, RPMI medium, methyl thiazolyl tetrazolium and Schneider's medium were purchased from Sigma-Aldrich (St. Louis, USA). Benznidazole was obtained from Roche (São Paulo, Brazil), and trimethoprim/sulfamethoxazole from Ducto (Goiás, Brazil). Trypsin-EDTA, RPMI 1640 medium without phenol red and gentamicin were purchased from Gibco (NY, USA). Silica gel 230-400 mesh from Merck (Darmstadt, Germany) was used for column chromatography, and silica gel Merck 60G was used for thin-layer chromatography. All solvents used were of PA and HPLC grade and purchased from Vetec (Brazil) and Sigma, respectively.

The 1D and 2D NMR spectra of acetogenins were performed on Brucker Avance DRX 400 spectrometers (Ettlingen, Germany) in CDCl3, containing 0.1% tetramethylsilane as the internal chemical shift standard. Electrospray Ionization Mass Spectrometry (ESI-MS) was performed using a Waters MICROMAS Q-TOF (Milford, Massachusetts, USA) equipped with an electrospray ion (ESI) source. A 200 µg mL–1solution of acetogenins in MeCN-H2O (1:1) was infused at 2 mL min–1, and the positive mass spectra was acquired with a m/z range between 50 and 1,000 daltons. The cone voltages were optimized for positive ion analysis in the range between 35 and 50 V. In the MS/MS experiments, the parent ion isolation width was 3.8 daltons, and the normalized collision energy was set at 30% for the compounds. Fifty scans were collected from 50 to 700 daltons to generate an average spectrum. Optical rotation was measured on a Perkin Elmer 341 polarimeter (Waltham, Massachusetts, USA). Final purification was performed on a Waters 501 apparatus (Milford, Massachusetts, USA) with a 486 UV-detector and a 746 integrator.

Plant Material

The fruits of Annona cornifolia A. St. -Hil. were collected from the Curvelo, Minas Gerais, Brazil, from January to March 1998. The species were identified by Dr. R. Mello-Silva and a voucher specimen (BHCB 68114) was deposited at the Instituto de Ciências Biológicas Herbarium, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.

Extraction and Isolation

Extraction of the dried and powdered seeds (850.0 g), by percolation (EtOH, 7 L, 50 h) gave a brown residue (120.0 g, F01), that was dissolved in MeOH/H2O (3:7) and successively extracted with C6H14 and CHCl3, resulting in 79.5, 7.3 and 21.1 g of hexane (F02), chloroform (F03) and hydroalcoholic (F04) fractions, respectively. Extract and fractions were evaporated to dryness before column chromatography and HPLC.

Acetogenins 3-7 and mixture 1+2 were isolated from F02, and acetogenins 8-15 from F03. The isolation of acetogenins 1-7 and 11-14 are described in previous publications by our group (Santos et al. 2006Santos LAR, Boaventura MAD and Pimenta LPS. 2006. Cornifolin, a new bis-tetrahydrofuran annonaceous acetogenin from Annona cornifolia. Biochem Syst Ecol 34: 78-82., Lima et al. 2009Lima LARS, Pimenta LPS and Boaventura MAD. 2009. Two new adjacent bis-tetrahydrofuran annonaceous acetogenins from seeds of Annona cornifolia. Planta Med 75: 80-83., 2010Lima LARS, Pimenta LPS and Boaventura MAD. 2010. Acetogenins from Annona cornifolia and their antioxidant capacity. Food Chem 122: 1129-1138.). F02 was chromatographed leading to 15.1 mg of a mixture of acetogenins 4-desoxylongimicin B + folianin A (1+2), 38.7 mg of squamocin M or isodesacetyluvaricin (3), 38.0 mg of squamocin L or desacetyluvaricin (4), 14.7 mg of folianin B (5), 40.0 mg of annofolin (6), and 6.0 mg of isolongimicin B (7). The acetogenins bullatacin or rolliniastatin-2 (11) (10.5 mg), asimicin or squamocin H (12) (3.5 mg), cornifolin (13) (30.4 mg), and annotacin (14) (12.0 mg) were obtained from F03. The isolation of acetogenins 8-10 and 15 are described below. Chloroform fraction (F03, 2.0 g) was fractionated by normal phase MPLC (with C6H14/ΦCH3/EtOAc/MeOH pure or in mixtures of increasing polarity), yielding 54 fractions of 75 mL each, combined according to their TLC patterns, yielding 10 groups of fractions. G-7 (620.0 mg, positive to Kedde reagent) underwent column chromatography (1.5 x 25 cm, 50.0 g SiO2 230-400 mesh, CH2Cl2/MeOH 97:3), yielding 52 fractions of 15 mL each, combined according to their TLC patterns in 19 subgroups of fractions. Subgroup 2 (45.4 mg) was submitted to a RP-HPLC (Shim-pack C18 5.0 µm, 20 x 250 mm cartridge column, flow rate 15 mL min–1, MeCN/H2O 70:30, detection 220 nm), leading to glaucanisin (8) (6.0 mg), to glaucanisin + parviflorin (8+9) (14.0 mg) and to glaucanisin + glaucanetin (8+10) (14.9 mg). Subgroup 7 (90.1 mg), from G-7, led to 15 subfractions, after being submitted to purification by RP-HPLC (Supelco SPLC-18 5.0 µm, 10 x 250 mm cartridge column, flow rate 2.5 mL min–1, MeCN/H2O 60:40, detection 220 nm). Subgroup 11 (84.7 mg), from G-7, by HPLC (same conditions as above) gave mixture of glaucanisin + longimicin B (8+15) (14.0 mg).

Cytotoxic Assay

The cytotoxic potential of acetogenins against the human melanoma (UACC-62), renal carcinoma (TK-10) and breast cancer (MCF-7) cell lines was evaluated in November 2001, using the sulforhodamine B (SRB) assay adopted by the National Cancer Institute, USA (Monks et al. 1991Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C, Langley J, Cronise P and Vaigro-Wolff A. 1991. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst 83: 757-766.). All cells were cultured in RPMI medium supplemented with 5% FBS and gentamicin, at 37°C with 5% CO2. Shortly before reaching confluence, the cells were detached with trypsin-EDTA and seeded into 96-well plates so that 100 µL in each well contained 10,000 UACC-62 and MCF-7 cells or 15,000 TK-10 cells. Acetogenins dissolved in DMSO 1% were tested firstly in triplicate at 20 µg mL–1, and after at different concentrations, acetogenins that inhibits 75% of growth human tumor cell lines. After 48 hours in the presence of the compound, the cells were fixed by adding 50 µL of cold 50% (w/v) trichloracetic acid to each well and incubating the plate at 4°C for 1 h. The supernatant was then discarded and the cells were washed five times with water. After drying at room temperature, 50 µL of SRB solution (0.4% w/v in 1% acetic acid) was added to each well, and the plate incubated for 30 min at 4°C. Unbound SRB was removed by washing five times with 1% acetic acid and the plates were dried at room temperature overnight. The plates were read at 515 nm after dissolution of the dye with Tris buffer (tris[hydroxymethyl] aminomethane). Etoposide was used as the positive control and 0.2% DMSO as the negative control. Each experiment was performed in triplicate. The concentration of the compound that produces 50% of growth inhibition (IC50, cytostatic effect) and the concentration of the compound that kills 50% of cells (LD50, cytocidal effect) were obtained from data of three independent experiments by non-linear regression using SOFTmax pro 5.3.

Antifungal Activity

Antifungal activity was evaluated in February 2004 and repeated in March 2011, with similar results. In vitro antifungal activity against Paracoccidioides brasiliensis, strains Pb-01 (ATCC- MYA-826), Pb-18 and Pb-18 virulent isolates (from the fungal collection of the Faculty of Medicine of the Universidade de São Paulo, SP, Brazil) were prepared in accordance with the guidelines in the CLSI document M27-A3 (CLSI 2008) and modified according to the suggestions of Johann et al. (2010)Johann S, Cisalpino PS, Watanabe GA, Cota BB, De Siqueira EP, Pizzolatti MG, Zani CL and De Resende MA. 2010. Antifungal activity of extracts of some plants used in the Brazilian traditional medicine against the pathogenic fungus Paracoccidioides brasiliensis. Pharm Biol 48: 388-396., to obtain a final suitable inoculum dilution for each strain. After homogenization by vortexing, the transmittance of the suspension was measured at wavelengths of 530 nm and adjusted to 69 to 70%.

The Minimal Inhibitory Concentration (MIC) was obtained from broth microdilution testing performed in accordance to described methods (CLSI 2008, Johann et al. 2010Johann S, Cisalpino PS, Watanabe GA, Cota BB, De Siqueira EP, Pizzolatti MG, Zani CL and De Resende MA. 2010. Antifungal activity of extracts of some plants used in the Brazilian traditional medicine against the pathogenic fungus Paracoccidioides brasiliensis. Pharm Biol 48: 388-396.). Amphotericin B and trimethoprim-sulfamethoxazole were included as positive antifungal control. Their stock solutions were prepared in DMSO and water, respectively.

The Minimal Fungicidal Concentration (MFC) values for acetogenins were determined as follows: from the microtiter plate used to determine the MIC values, the test wells that showed: a) complete fungal growth inhibition (clear wells), b) growth similar to that of the no-drug control well, and c) growth control wells, were selected for the assay to determine the MFC. The MFC was determined as the lowest drug concentration at which fewer than three colonies were able to grow (Espinel-Ingroff et al. 2001Espinel-Ingroff A, Boyle K and Sheehan DJ. 2001. In vitro antifungal activities of voriconazole and reference agents as determined by NCCLS methods: review of the literature. Mycopathologia 150: 101-115.).

Trypanocidal Activity

In vitro assay with amastigote and trypomastigote forms of Trypanosoma cruzi was performed in October 2003 and repeated in June 2013, with similar results, according to protocols established by Buckner et al. (1996)Buckner FS, Verlinde CL, La Flamme AC and Van Voorhis WC. 1996. Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing β-galactosidase. Antimicrob Agents Chemother 40: 2592-2597. with modifications (Romanha et al. 2010). Briefly, parasites and culture procedures: T. cruzi (Tulahuen strain) expressing the Escherichia coli β-galactosidase gene were grown on monolayer of mouse L929 fibroblasts. Cultures to be assayed for β-galactosidase activity were grown in RPMI 1640 medium (pH 7.2–7.4) without phenol red plus 10% foetal bovine serum and glutamine. For the bioassay, 96 well tissue culture microplates were seeded with L929 fibroblasts in 80 µL with a density of 4.0 x 103fibroblasts per well and incubated overnight at 37°C and 5% CO2. β-galactosidase-expressing trypomastigotes in 20 µL of medium were then added at a density of 4.0 x 104 per well. After 2 h of contact, the medium with trypomastigotes that had not penetrated the cells was discarded and replaced by 200 µL of fresh medium. After 48 h, the medium was discarded again and replaced by 180 µL of fresh medium and 20 µL of a solution of acetogenins at concentration of 20 µg mL–1. Each compound was tested in triplicate. After seven days of culture development, chlorophenol red β-D-galactopyranoside at 100 µM and Nonidet P-40 at 0.1% were added to the plates and the plates were then incubated overnight at 37°C. The absorbance was measured at 570 nm in an automated microplate reader. Benznidazole at its half maximal inhibitory concentration (IC50) (1 µg/mL = 3.8 µM) was used as a positive control. The results were expressed as the percentage of parasite growth inhibition.

Cytotoxic test for determination of the Selectivity Index (SI) was performed in June 2013. In vitro cytotoxic test was also carried out to determine the toxicity of the compounds over L929 cells by alamarBlue®. The same cell number, time of the cells development and time of compound exposure were used for the β-galactosidase assay. After 96 hours of compounds exposure the alamarBlue® was added and the absorbance at 570 and 600 nm was measured after 4-6 h. The cell viability was expressed as the percentage of difference in the reduction between treated and untreated cells (Romanha et al. 2010). IC50 values were calculated by linear interpolation and the Selectivity Index (SI) was determined based on the ratio of the IC50 value in the host cell divided by the IC50 value of the parasite (IC50/IC50 ratio).

Leishmanicidal Activity

Assays with amastigote-like forms of Leishmania (Leishmania) amazonensis were performed in September 2003, using the MTT (methyl thiazolyl tetrazolium)-based colorimetric assay (Callahan et al. 1997Callahan HL, Portal AC, Devereaux R and Grogl M. 1997. An axenic amastigote system for drug screening. Antimicrob Agents Chemother 41: 818-822.). Amphotericin B was used as the positive control. Promastigotes forms of L. amazonensis (strain IFLA/BR/196/PH-8) were obtained from lesions of experimentally infected hamsters. The parasites were incubated for 9 days at 26°C in Schneider's medium, buffered at pH 7.2. The promastigotes forms were then stimulated to differentiate into amastigote-like forms by rising the incubation temperature to 32°C and lowering the pH of the medium to 6.0. After 7 days under these conditions, 90% of the parasites differentiated. The parasite concentration was adjusted to 1 x 108cells mL–1, and 90 µL were added to each well of 96-well plates, followed by 10 µL of the solutions containing the samples (20 µg mL–1) and control drug (0.2 µg mL–1 amphotericin B). Only compounds that caused at least 75% of inhibition were tested again in different concentration. The plates were incubated at 32°C for 72 h and the number of parasites was estimated using the MTT based colorimetric assay. The LD50(lethal dose that kills 50% of cells) was obtained from data of three independent experiments by non-linear regression using the SOFTmax pro 5.3.

Statistical Analyses

Measurements were conducted in triplicate. All the data are shown as mean standard deviation (SD). The values of p < 0.05 were considered statistically significant.

RESULTS AND DISCUSSION

The identification (by IR, 1H and 13C NMR and MS spectra) of acetogenins 1-2, 5-7, and 13-14, isolated for the first time from Annona cornifolia, are described in previous publications by our group (Santos et al. 2006Santos LAR, Boaventura MAD and Pimenta LPS. 2006. Cornifolin, a new bis-tetrahydrofuran annonaceous acetogenin from Annona cornifolia. Biochem Syst Ecol 34: 78-82., Lima et al. 2009Lima LARS, Pimenta LPS and Boaventura MAD. 2009. Two new adjacent bis-tetrahydrofuran annonaceous acetogenins from seeds of Annona cornifolia. Planta Med 75: 80-83., 2010Lima LARS, Pimenta LPS and Boaventura MAD. 2010. Acetogenins from Annona cornifolia and their antioxidant capacity. Food Chem 122: 1129-1138.). Spectrometric data of the known acetogenins (new for this specie) 3-4 and 11-12 are described by Lima et al. (2010)Lima LARS, Pimenta LPS and Boaventura MAD. 2010. Acetogenins from Annona cornifolia and their antioxidant capacity. Food Chem 122: 1129-1138.. The acetogenins squamocin L (4) and squamocin M (3) were isolated by Sahai et al. (1994). The squamocin L was identical with desacetyluvaricin, isolated from Uvaria accuminata as well as Annona glabra seeds. The squamocin M was identical with isodesacetyluvaricin, isolated from Uvaria narum (Sahai et al. 1994, Zeng et al. 1996Zeng L, Ye Q, Oberlies NH, Shi G, Gu ZM, He K and McLaughlin JL. 1996. Recent advances in annonaceous acetogenins. Nat Prod Rep 3: 275-306., Cavé et al. 1997Cavé A, Figadère B, Laurens A and Cortes D. 1997. Acetogenins from annonaceae. In: CAVÉ A, CORTES D, FIGADÈRE B, LAURENS A AND PETTIT GR. Progress in the chemistry of organic natural products, New York: Springer-Wien, p. 81-289.). Acetogenins 8-10 and 15 were compared to other molecules in the literature: glaucanisin (8)(Waechter et al. 1995Waechter AI, Hocquemiller R, Laurens A and Cavé A. 1995. Glaucanisin, a new acetogenin from Annona glauca. Nat Prod Lett 6: 133-138.), parviflorin or squamocin E (9) (Gallardo et al. 1998Gallardo T, Aragon R, Tormo JR, Blazquez MA, Zafra-Polo MC and Cortes D. 1998. Acetogenins from Annona glabra seeds. Phytochemistry 47: 811-816.), glaucanetin (10) (Waechter et al. 1997Waechter AI, Hocquemiller R, Laurens A and Cavé A. 1997. Glaucanetin and 10-hydroxy-glaucanetin, two new acetogenins from Annona glauca. Nat Prod Lett 10: 205-212.) and longimicin B (15) (Ye et al. 1996Ye Q, He K, Oberlies NH, Zeng L, Shi G, Evert D and McLaughlin JL. 1996. Longimicins A-D: novel bioactive acetogenins from Asimina longifolia (Annonaceae) and structure-activity relationships of asimicin type of annonaceous acetogenins. J Med Chem 39: 1790-1796.).

The cytotoxic activities of acetogenins 1-15 at 20 µg mL–1 are shown in Table I. IC50 and LD50 were established only for those that inhibited 75% of the cells at the concentration of 20 µg mL–1 (Table II). These acetogenins exhibited significant cytotoxic activity and selectivity for the tumor cell lines, being more active against MCF-7. Annofolin (6) presented lower LD50 values than the etoposide for all the lines tested, while annofolin (6), isolongimicin B (7) and the mixtures 8+9, 8+10 and 8+15showed lower IC50 values than the etoposide for the TK-10 cell line. The mixtures showed higher cytostatic activity than pure glaucanisin (8) against TK-10 and UACC-62 cells, suggesting a potentiation of the effect by the two acetogenins.

TABLE I
In vitro effect of acetogenins from Annona cornifolia, tested at 20 µg mL–1, against human cancer cell lines, Trypanosoma cruzi and Leishmania amazonensis.
TABLE II
In vitro cytotoxicity, trypanocidal and leishmanicidal activities of acetogenins from Annona cornifolia.

Yang et al. (2009)Yang H, Zhang N, Li X, Chen J and Cai B. 2009. Structure-activity relationships of diverse annonaceous acetogenins against human tumor cells. Bioorg Med Chem Lett 19: 2199-2202.established that (i) adjacent bis-THF acetogenins are the most potent antitumor agents among this class of compounds, (ii) if all other structural features are identical, C-35 acetogenins are more active than those with 37 carbon atoms; (iii) for better activity, the distance between the OH-flanked THF and the γ-lactone must be 13 carbon atoms, and (iv) acetogenins with stereochemical arrangement of threo/trans/threo/trans/threo around THF rings are less potent than those with stereochemical arrangement of threo/trans/threo/trans/erythro. However, annofolin (6), the most active agent in this work, is interestingly a C-37 acetogenin with 9 carbon atoms between the OH-flanked THF and the γ-lactone, presenting a stereochemical arrangement of erythro/trans/threo/trans/threo around THF rings.

The ten pure and the four mixtures of acetogenins were tested against three isolates of Paracoccidioides brasiliensis (Pb01, Pb18 and Pb18 virulent) at 1.17-150 µg mL–1, their Minimal Inhibitory Concentration (MIC) and Minimal Fungicidal Concentration (MFC) being determined. MIC and MFC values were found to be equal and are presented in Table III. Acetogenins 5-12 showed antifungal activity at concentrations ≤ 150 µg mL–1, thus being more active than trimethoprim-sulfamethoxazole, the first class of drugs used to treat paracoccidioidomycosis, but less active than amphotericin B. Cornifolin (13) was inactive at this concentration against all three isolates. Annofolin (6), isolongimicin B (7) and bullatacin (11) showed the highest levels of activity against the three isolates of P. brasiliensis. Pb18 exhibited some selectivity, being more sensitive to squamocin M (3) and folianin B (5).

TABLE III
In vitro antifungal activity of acetogenins from Annona cornifolia against Paracoccidioides brasiliensis.

There are few studies in the literature reporting the activity of natural products against the pathogenic fungus P. brasiliensis. The natural product (R)-goniothalamin and its synthetic enantiomer were evaluated against a panel of microorganisms that included three strains of P. brasiliensis (Pb-01, Pb-18 and Pb-B339). Their MIC values were in the range between 7 and 22 µg mL–1 for P. brasiliensis (Fátima et al. 2008Fátima A, Martins CVB, De Resende MA, Ferreira Magalhães TF, Santiago Lima BH, Watanabe GA, Gois Ruiz ALT, de Carvalho JE and Pilli RA. 2008. Antifungal activity of goniothalamin enantiomers. Lett Drug Des Discov 5: 74-78.). In this study, annofolin (6) and isolongimicin B (7) showed higher activity than the goniothalamin enantiomers against P. brasiliensis. In the experiment carried out by Lima et al. (2011)Lima LARS, Johann S, Cisalpino PS, Pimenta LPS and Boaventura MAD. 2011. Antifungal activity of 9-hydroxy-folianin and sucrose octaacetate from the seeds of Annona cornifolia A. St. -Hil. (Annonaceae). Food Res Int 44: 2283-2288., the authors tested the in vitrosusceptibility of twelve clinical P. brasiliensis isolates to 9-hydroxy-folianin, an adjacent bis-THF acetogenin. The results demonstrated that all isolates were susceptible to 9-hydroxy-folianin, with a MIC of 3.4 to 27.7 µg mL–1. In our study, annofolin (6) was more active than 9-hydroxy-folianin against the three isolates of P. brasiliensis.

The acetogenins, at 20 µg mL–1, were also tested against the amastigote and trypomastigote forms of T. cruzi. The results are expressed in percentages of parasite growth inhibition (Table I). Squamocin M (3), annofolin (6), annotacin (14) and the mixtures (1+2), (8+9) and (8+10) inhibited more than 50% of amastigote and trypomastigote forms of T. cruzi; their IC50 values are given in Table II.

All the acetogenins showed similar or greater trypanocidal effects as compared to benznidazole (IC50 values less 1.7 µM), but showed toxicity at the same concentration, causing 50% of cell death (L929 cells). Thereby, no selective toxicity to the parasites was observed (Selectivity Index, SI = 1) when a tissue culture assay was used. The acetogenins 3, 6, 14 and the mixtures (1+2), (8+9) and (8+10) showed growth inhibition of the parasites in the range of 51.9-100.0% (Table I). This level of activity is considered to be significant (Croft et al. 1988Croft SL, Walker JJ and Gutteridge WE. 1988. Screening of drugs for rapid activity against Trypanosoma cruzi trypomastigotes in vitro. Trop Med Parasitol 39: 145-148.). Some studies confirmed the trypanocidal activity of the acetogenins against T. cruzi parasites (Waechter et al. 1998Waechter A, Yaluff G, Inchausti A, Rojas de Arias A, Hocquemiller R, Cavé A and Fournet A. 1998. Leishmanicidal and trypanocidal activities of acetogenins isolated from Annona glauca. Phytother Res 12: 541-544., González-Coloma et al. 2002González-Coloma A, Guadaño A, De Inés C, Martínez-Díaz R and Cortes D. 2002. Selective action of acetogenin mitochondrial complex I inhibitors. Z Naturforsch C 57: 1028-1034.).

The activity of all acetogenins against L. amazonensisamastigotes was evaluated (also at 20 µg mL–1). Those that presented inhibition higher than 75% had their LD50values determined (Table II). Annofolin (6) and annotacin (14), both C-37 bis-THF acetogenins, were the most active, presenting LD values of 6.4 x 101 and 7.2 x 101 µM, respectively, indicating the relevance of these compounds in the search for new leishmanicidal drugs.

Few works report the leishmanicidal activity of acetogenins with one or two THF rings against the promastigote and amastigote forms of Leishmania (Waechter et al. 1998Waechter A, Yaluff G, Inchausti A, Rojas de Arias A, Hocquemiller R, Cavé A and Fournet A. 1998. Leishmanicidal and trypanocidal activities of acetogenins isolated from Annona glauca. Phytother Res 12: 541-544., Raynaud-Le Grandic et al. 2004, Vila-Nova et al. 2011Vila-Nova NA, Morais SM, Falcão MJC, Machado LKA, Beviláqua CML, Costa IRS, Brasil NVGPS and Andrade Júnior HF. 2011. Leishmanicidal activity and cytotoxicity of compounds from two Annonaceae species cultivated in Northeastern Brazil. Rev Soc Bras Med Trop 44: 567-571.). According to Raynaud-Le Grandic et al. (2004), the stereostructural arrangement of threo/trans/threo/trans/erythro around the THF ring favors leishmanicidal activity. Annofolin (6), with the stereostructural arrangement erythro/trans/threo/trans/threo, exerted the highest level of leishmanicidal activity among the acetogenins tested in this work.

CONCLUSIONS

Although the cytotoxic activity of acetogenins has already been described, the high level of this activity observed in these compounds confirms their potential for being used in anti-cancer therapy. Regarding leishmanicidal and trypanocidal activities, an inhibition of 87% of L. amazonensis amastigotes and 100% of T. cruziamastigotes and trypomastigotes was observed, when tested at the concentration of 20 µg mL–1. Moreover, six acetogenins showed more activity against all the three tested isolates of P. brasiliensis than trimethoprim-sulfamethoxazole, a drug used for treating paracoccidioidomycosis. Thus, acetogenins hold great promise in treating a number of diseases that have a huge impact on millions of people worldwide.

We are grateful to Dr. Renato Mello-Silva for the botanical identification of this plant species.

REFERENCES

  • Buckner FS, Verlinde CL, La Flamme AC and Van Voorhis WC. 1996. Efficient technique for screening drugs for activity against Trypanosoma cruzi using parasites expressing β-galactosidase. Antimicrob Agents Chemother 40: 2592-2597.
  • Callahan HL, Portal AC, Devereaux R and Grogl M. 1997. An axenic amastigote system for drug screening. Antimicrob Agents Chemother 41: 818-822.
  • Castillo-Garit JA, Abad C, Rodrígues-Borges JE, Marrero-Ponce Y and Torrens F. 2012. A review of QSAR studies to discover new drug-like compounds against leishmaniasis and trypanosomiasis. Curr Top Med Chem 12: 852-865.
  • Cavé A, Figadère B, Laurens A and Cortes D. 1997. Acetogenins from annonaceae. In: CAVÉ A, CORTES D, FIGADÈRE B, LAURENS A AND PETTIT GR. Progress in the chemistry of organic natural products, New York: Springer-Wien, p. 81-289.
  • CLSI - Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of yeasts: approved standard - Third Ed. CLSI document M27-A3. CLSI, Wayne, PA, USA.
  • Correa MP. 1984. Dicionário de plantas úteis do Brasil e de plantas exóticas cultivadas, Rio de Janeiro: Imprensa Nacional 1: 194.
  • Croft SL, Walker JJ and Gutteridge WE. 1988. Screening of drugs for rapid activity against Trypanosoma cruzi trypomastigotes in vitro. Trop Med Parasitol 39: 145-148.
  • Espinel-Ingroff A, Boyle K and Sheehan DJ. 2001. In vitro antifungal activities of voriconazole and reference agents as determined by NCCLS methods: review of the literature. Mycopathologia 150: 101-115.
  • Fátima A, Martins CVB, De Resende MA, Ferreira Magalhães TF, Santiago Lima BH, Watanabe GA, Gois Ruiz ALT, de Carvalho JE and Pilli RA. 2008. Antifungal activity of goniothalamin enantiomers. Lett Drug Des Discov 5: 74-78.
  • Gallardo T, Aragon R, Tormo JR, Blazquez MA, Zafra-Polo MC and Cortes D. 1998. Acetogenins from Annona glabra seeds. Phytochemistry 47: 811-816.
  • González-Coloma A, Guadaño A, De Inés C, Martínez-Díaz R and Cortes D. 2002. Selective action of acetogenin mitochondrial complex I inhibitors. Z Naturforsch C 57: 1028-1034.
  • Johann S, Cisalpino PS, Watanabe GA, Cota BB, De Siqueira EP, Pizzolatti MG, Zani CL and De Resende MA. 2010. Antifungal activity of extracts of some plants used in the Brazilian traditional medicine against the pathogenic fungus Paracoccidioides brasiliensis. Pharm Biol 48: 388-396.
  • Lima LARS, Johann S, Cisalpino PS, Pimenta LPS and Boaventura MAD. 2011. Antifungal activity of 9-hydroxy-folianin and sucrose octaacetate from the seeds of Annona cornifolia A. St. -Hil. (Annonaceae). Food Res Int 44: 2283-2288.
  • Lima LARS, Pimenta LPS and Boaventura MAD. 2009. Two new adjacent bis-tetrahydrofuran annonaceous acetogenins from seeds of Annona cornifolia. Planta Med 75: 80-83.
  • Lima LARS, Pimenta LPS and Boaventura MAD. 2010. Acetogenins from Annona cornifolia and their antioxidant capacity. Food Chem 122: 1129-1138.
  • Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C, Langley J, Cronise P and Vaigro-Wolff A. 1991. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst 83: 757-766.
  • Newman DJ, Cragg GM and Snader KM. 2003. Natural products as sources of new drugs over the period 1981-2002. J Nat Prod 66: 1022-1037.
  • Pettit GR, Mukku VJRV, Cragg G, Herald DL, Knight JC, Herald CL and Chapuis JC. 2008. Antineoplastic agents. 558. Ampelocissus sp. Cancer cell growth inhibitory constituents. J Nat Prod 71: 130-133.
  • Pimenta LPS, Nascimento FC, Assunção ACS and Boaventura MAD. 2003. Acetogeninas de anonáceas isoladas de folhas de Rollinia laurifolia. Quím Nova 26: 319-322.
  • Pimenta LPS, Nascimento FC, Assunção ACS, Oliveira AB and Boaventura MAD. 2001. Laurifolin, a novel acetogenin from Rollinia laurifolia leaves. Tetrahedron Lett 42: 8433-8434.
  • Pimenta LPS, Nascimento FC and Boaventura MAD. 2005. Acetogenins from the leaves of Rollinia laurifolia. Helv Chim Acta 88: 3225-3231.
  • Raynaud-LE Grandic S, Fourneau C, Laurens A, Bories C, Hocquemiller R and Loiseau PM. 2004. In vitro antileishmanial activity of acetogenins from Annonaceae. Biomed Pharmacother 58: 388-392.
  • Romanha AJ et al. 2010. In vitro and in vivo experimental models for drug screening and development for Chagas disease. Mem Inst Oswaldo Cruz 105: 233-238.
  • Sahai M et al. 1994. Annonaceous acetogenins from the seeds of Annona squamosa, adjacent bis-tetrahydrofuranic acetogenins. Chem Pharm Bull 42: 1163-1174.
  • Santos LAR, Boaventura MAD and Pimenta LPS. 2006. Cornifolin, a new bis-tetrahydrofuran annonaceous acetogenin from Annona cornifolia. Biochem Syst Ecol 34: 78-82.
  • Santos LAR, Boaventura MAD and Pimenta LPS. 2007. Acetogeninas de anonáceas bioativas isoladas das sementes de Annona cornifolia A. St. -Hil. Rev Bras Plantas Med 9: 48-51.
  • Santos LP, Boaventura MAD and Oliveira AB. 1994. Crassiflorina, uma acetogenina tetra-hidrofurânica citotóxica de Annona crassiflora (araticum). Quím Nova 17: 387-391.
  • Santos LP, Boaventura MAD, Sun NJ, Cassady JM and Oliveira AB. 1996. Araticulin, a new bis-tetrahydrofuran polyketide from Annona crassiflora seeds. Phytochemistry 42: 705-707.
  • Shikanai-Yasuda MA, Telles Filho FQ, Mendes RP, Colombo AL and Moretti ML. 2006. Guideliness in paracoccidioidomycosis. Rev Soc Bras Med Trop 39: 297-310.
  • Vila-Nova NA, Morais SM, Falcão MJC, Machado LKA, Beviláqua CML, Costa IRS, Brasil NVGPS and Andrade Júnior HF. 2011. Leishmanicidal activity and cytotoxicity of compounds from two Annonaceae species cultivated in Northeastern Brazil. Rev Soc Bras Med Trop 44: 567-571.
  • Waechter AI, Hocquemiller R, Laurens A and Cavé A. 1995. Glaucanisin, a new acetogenin from Annona glauca. Nat Prod Lett 6: 133-138.
  • Waechter AI, Hocquemiller R, Laurens A and Cavé A. 1997. Glaucanetin and 10-hydroxy-glaucanetin, two new acetogenins from Annona glauca. Nat Prod Lett 10: 205-212.
  • Waechter A, Yaluff G, Inchausti A, Rojas de Arias A, Hocquemiller R, Cavé A and Fournet A. 1998. Leishmanicidal and trypanocidal activities of acetogenins isolated from Annona glauca. Phytother Res 12: 541-544.
  • Yang H, Zhang N, Li X, Chen J and Cai B. 2009. Structure-activity relationships of diverse annonaceous acetogenins against human tumor cells. Bioorg Med Chem Lett 19: 2199-2202.
  • Ye Q, He K, Oberlies NH, Zeng L, Shi G, Evert D and McLaughlin JL. 1996. Longimicins A-D: novel bioactive acetogenins from Asimina longifolia (Annonaceae) and structure-activity relationships of asimicin type of annonaceous acetogenins. J Med Chem 39: 1790-1796.
  • Zeng L, Ye Q, Oberlies NH, Shi G, Gu ZM, He K and McLaughlin JL. 1996. Recent advances in annonaceous acetogenins. Nat Prod Rep 3: 275-306.

Publication Dates

  • Publication in this collection
    June 2014

History

  • Received
    8 Feb 2013
  • Accepted
    11 Nov 2013
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