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

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. 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.


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 1984).Annonaceous acetogenins, isolated exclusively from the Annonaceae family until 2008 (Pettit et al. 2008), exhibit a broad range of biological properties (Yang et al. 2009).Our group has already investigated acetogenins from the seeds of A. crassiflora (Santos et al. 1994(Santos et al. , 1996) ) and A. cornifolia (Santos et al. 2006, 2007, Lima et al. 2009, 2010), as well as from the leaves of Rollinia laurifolia (Pimenta et al. 2001(Pimenta et al. , 2003(Pimenta et al. , 2005)).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 1984).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. 2012).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. 2006).In the absence of drug therapy, paracoccidioidomycosis is usually fatal (Shikanai-Yasuda et al. 2006, Johann et al. 2010).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. 2003).

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 CDCl 3 , 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 -1 solution of acetogenins in MeCN-H 2 O (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 BIOLOGICAL ACTIVITIES OF Annona cornifolia

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. 1991).All cells were cultured in RPMI medium supplemented with 5% FBS and gentamicin, at 37°C with 5% CO 2 .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 (IC 50 , cytostatic effect) and the concentration of the compound that kills 50% of cells (LD 50 , cytocidal effect) were obtained from data of three independent experiments by nonlinear regression using SOFTmax pro 5.3.2010), 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. 2010).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. 2001).

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) 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 10 3 fibroblasts per well and incubated overnight at 37°C and 5% CO 2 .β-galactosidase-expressing LUCIANA A.R.S. LIMA et al. trypomastigotes in 20 µL of medium were then added at a density of 4.0 x 10 4 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-Dgalactopyranoside 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 (IC 50 ) (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).IC 50 values were calculated by linear interpolation and the Selectivity Index (SI) was determined based on the ratio of the IC 50 value in the host cell divided by the IC 50 value of the parasite (IC 50 /IC 50 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. 1997).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 10 8 cells 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 LD 50 (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.
The cytotoxic activities of acetogenins 1-15 at 20 µg mL -1 are shown in Table I.IC 50 and LD 50 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 LD 50 values than the etoposide for all the lines tested, while annofolin (6), isolongimicin B (7) and the mixtures 8+9, 8+10 and 8+15 showed lower IC 50 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.Yang et al. (2009) 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.
The activity of all acetogenins against L. amazonensis amastigotes was evaluated (also at 20 µg mL -1 ).Those that presented inhibition higher than 75% had their LD 50 values 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 10 1 and 7.2 x 10 1 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. 1998, Raynaud-Le Grandic et al. 2004, Vila-Nova et al. 2011).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. 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 hold great promise in treating a number of diseases that have a huge impact on millions of people worldwide.
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. (

TABLE II In vitro cytotoxicity, trypanocidal and leishmanicidal activities of acetogenins from Annona cornifolia.
concentration that kills 50% of the cells or parasites (cytocidal effect); c Selectivity Index (SI) is the ratio of IC 50 on normal cell line to the IC 50 on parasite; d positive control.nt: not tested *p < 0.05 compared with positive control.BIOLOGICAL ACTIVITIES OF Annona cornifolia a concentration that inhibits 50% of growth (cytostatic effect); b

TABLE III In vitro antifungal activity of acetogenins from Annona cornifolia against Paracoccidioides brasiliensis.
a MIC: Minimal Inhibition Concentration; MFC: Minimal Fungicidal Concentration; b positive control.nd: no detected activity at 150.0 µg mL -1 .*p < 0.05 compared with positive control.