Antioxidant, antibacterial, leishmanicidal and trypanocidal activities of extract and fractions of Manilkara rufula stem bark

Manilkara rufula belongs to family Sapotaceae and has received little attention regarding its pharmacological properties and chemical composition. Therefore, our objective was to determine the farmacological activities and preliminary chemical profile of the stem bark from M. rufula. The plant stem bark of M. rufula was collected and ethanolic extract and fractions were prepared. The antioxidant activity was determined by •DPPH and ABTS•+ scavenger methods and inhibition of β-carotene oxidation. The minimum inhibitory and bactericidal concentrations were estimated through the broth microdilution method against gram positive and negative bacteria. Cytotoxicity, antioxidant and cytoprotection potential were investigated on culture macrophages. Antiproliferative activity was evaluated by Alamar Blue method. The in vitro arginase activity from Leishmania amazonensis was determined in presence of the samples. In addition, their effects on survival of L. amazonensis and Trypanosoma cruzi amastigotes in mice macrophages were assessed. The preliminary phytochemical profile was evaluated by qualitative methods of classical phytochemistry, infrared spectroscopy, LC-MS/MS and CG-MS. The samples presented antioxidant, bactericidal, leishmanicidal and trypanocidal properties without toxic characteristics on normal cells. Triterpenes were observed in the hexane fraction, while glycosides, aromatic rings, fatty esters, proanthocyanidin dimers and trimers, catechin and various terpenes were observed in the other fractions. Keywords— Sapotaceae, Manilkara rufula, pharmacological activities, chemical composition.

37ºC. After 30 min, the absorbance of samples was measured at 750 nm. The amount of total phenols in samples was determined based on the standard curve of gallic acid (0.5 at 25 μg; Sigma-Aldrich, St. Louis, MO, USA) and expressed as equivalent μg of gallic acid per milligram of sample (μg GAE/mg). The calibration curve equation for gallic acid was y= 0.066x + 0.0651, R 2 = 0.9805.

D. Qualitative phytochemical study
Extract and fractions of the stem bark of M. rufula were analyzed by thin layer chromatography for qualitative identification of alkaloids and flavonoids, with specific developers for each class. The samples were previously solubilized in methanol, except for the hexane fraction, which was solubilized in chloroform. They were then plated with silica gel (stationary phase) and eluted in a glass vat containing an ethyl acetate: methanol (8:2, Synth, Sao Paulo, Brazil) mixture. The plates were observed under UV light 254 nm and 365 nm and then sprayed with the NP-PEG (1% diphenylboryloxyethylamine in methanol, followed by 5% polyethylene glycol 4000 solution in ethanol) and Dragendorff reagents for discovery of flavonoids and alkaloids, respectively (Wagner and Blad, 2009). The presence of triterpenes was evaluated by Liebermann -Burchard method. About 2 mg of extract and fraction s were diluted in 2 mL of chloroform (Synth, Sao Paulo, Brazil). Then, 4 mL of acetic anhydride (Synth, Sao Paulo, Brazil) and 4 drops of sulfuric acid (Synth, Sao Paulo, Brazil) were added. The presence of triterpenes wasindicated by the change from blue to green (Matos, 1997).

E. Infrared spectroscopy
Infrared analyzes were performed using the Perkin Elmer spectrophotometer model Spectrum Two ATR-FTIR, with horizontal attenuated total reflectance accessory employing a zinc selenide crystal. The spectra were obtained by spreading the sample onto the crystal surface of ATR. For each analysis, the cell was cleaned with acetone (Synth, Sao Paulo, Brazil). All spectra were obtained in the region of 4000 to 500 cm -1 , with resolution of 4 cm -1 and 32 scans (Ruschel et al., 2014).

F. LC-MS/MS
Chromatographic analyses of samples were performed using a UPLC Acquity chromatograph coupled with a TQD Acquity mass spectrometer (Micromass -Waters), with an electrospray ionization (ESI) source in the negative mode. The column was a Phenomenex Luna C-18 (250x4.6 mm, 5 µm). The mobile phases were foram water/formic acid 0.1% (phase A) e acetonitrile/formic acid 0.1% (phase B). The flow rate was 1 mL/min with a linear gradient starting at 0% B and increasing to up 100% in 60 min, before holding until 5 min, and then returning to initial conditions, followed by column re-equilibration. The ESI conditions were: capillary = 4.5 kV, cone = 30 V, source temperature = 300 °C, desolvation temperature = 300 °C, and collision energy = 30 V, with data acquisition between m/z 50 and 1000. The components of M. rufula samples were putatively identified by comparing their m/z values and fragmentation patterns with previous reports.

G. CG-MS
The identification of the compounds in the hexane fraction from M. rufula stem bark was performed after separation by high resolution gas chromatography with the capillary column "Rtx -5MS" -30 m (length) x 0.25 mm (internal diameter) x 0.25 µm (film thickness) nominal, helium gas as drag gas coupled to a mass detector (CG-MS Model QP2020, Shimadzu, Kyoto, Japan). The identification of compounds was performed by comparing the mass spectra of the samples with those found in NIST version 14.

J. Citoprotective and antioxidant activities
Macrophages (1x10 5 well -1 ) derived from THP-1 cell line were placed into a 96-well plate and pre-incubated at 37 °C with extract and fractions of M. rufula (50 µg mL -1 ). For the cytoprotective assay, cells were washed twice in phosphate-buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA) 24 h later. The medium was replaced, and H2O2 (Synth, Sao Paulo, Brazil) (1 mM) was added, following incubation for 18 h. The residual cell viability was determined as described above (Facundo et al., 2007).
To determine the antioxidant activity of isolated extract and fractions in cells, the same procedure was performed. After incubation with H2O2, the cells were washed in PBS, incubated in 2',7'-dichlorofluorescein diacetate (DCFH-DA; Molecular Probes, Eugene, OR, USA) at 10 µM for 1 h at 37°C in a dark chamber. The extracellular DCFH-DA was removed after washing the cells twice in PBS. The oxidation of DCFH was determined by fluorescence (ex=485 nm; ex=520 nm) using a microplate reader, since the fluorescent signal indicates the intracellular redox state. In both assays, ethanol (5%) and tempol (10 µM) were selected as negative and positive controls, respectively (Jeong et al., 2009). K. Hemeoxigenase activuty Cells (5x10 5 well -1 ) were pre-incubated for 24 h with fractions at 50 µg mL -1 or tempol (10 µM). Afterwards, the harvested cells were subjected to three cycles of freezethawing before addition to a reaction mixture consisting of phosphate buffer (1 ml final volume, pH 7.4) containing magnesium chloride (2 mM), NADPH (0.8 mM), glucose-6-phosphate (2 mM), glucose-6-phosphate dehydrogenase (0.2 U), rat liver cytosol as a source of biliverdin reductase, and the substrate hemin (20 μM). The reaction mixture was incubated in the dark at 37 °C for 1 h and was terminated by the addition of 1 mL of chloroform. After being vigorously vortexed and centrifuged, the extracted bilirubin in the chloroform layer was measured by the difference in absorbance between 464 and 530 nm (ε = 40 mM −1 ·cm −1 ) (Jeong et al., 2009).

L. Suscetibility testing
The antibacterial activity of EEMR and fractions were examined by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), according to the Institute of Clinical and Laboratory Standards (Santos et al., 2017). To determine the MIC, 5×10 5 CFU mL -1 diluted in brain heart infusion medium (Difco, USA) were incubated with EEMR and fractions (1-1000 μg mL -1 ) in 96-well microplates for 24 h at 37 °C. The vehicle control was ethanol (5%). MIC was defined as the lowest concentration of EEMR or fraction that allowed no visible growth after incubation with 0.01% resazurin dye (Sigma-Aldrich, St. Louis, MO, USA) for 60 min at room temperature. MBC was determined by sub-culturing 10 µL of each incubated well that had a concentration higher than the MIC on Müller-Hinton agar. The MBC was then treated as the lowest concentration of each sample with no visible colony growth on agar plates.
The bacterial strains used in this study were S. aureus The reaction mixtures were incubated in a 37 °C water bath for 15 min. The quantity of the enzyme used was adjusted to 10% of the maximum consumption of L-arginine substrate. Quantification of urea production was performed by the method described by Berthelot (Fawcett and Scott, 1960;Manjolin et al., 2013). Briefly, arginase catalytic capacity was stopped by transferring 10 µL of the reaction mixture into 750 µL of reagent A (20 mM phosphate buffer pH 7, containing 60 mM salicylate, 1 mM sodium nitroprusside and 500 IU of uréase; Labclin, Sao Paulo, Brazil). This mixture was incubated at 37 °C for 5 min. Next, 750 µL of reagent B (10 mM sodium hypochlorite and 150 mM NaOH; Labclin) was added, and the samples were incubated at 37 °C for 10 min. The absorbance was measured at at 600 nm using a GEHAKA 340G spectrophotometer. The positive and negative controls were performed under the same conditions in the absence of inhibitor. The experiments were performed in triplicate in at least two independent experiments.

O. Leishmanicidal activity
Peritoneal exudate macrophages were obtained by injection ofthioglycolate (3%) into the peritoneal cavity of Briefly, the exudate cells were recovered by peritoneal lavage with sterile saline. Cell suspension was then centrifuged at 1500 rpm for 10 min at 4 °C and the pellet was resuspended in DMEM medium (Sigma-Aldrich, St. Louis, MO, USA). After counting in a Newbauer camera, cells were plated in 96-well plates (5x10 4 /well) in DMEM medium and incubated for 16 h at 37 °C under CO2 atmosphere (5%). Plates were then washed three times with warm saline to remove non-adherent cells. The macrophages were infected with stationary growth phase promastigotes of L. amazonensis at a ratio of 5:1 macrophage. The co-culture was then incubated at 35°C under CO2 atmosphere (5%) during 24 h, followed by washing to remove the non-internalized parasites. Infected macrophages were incubated with the plant extract and fractions (20 μg mL -1 ) in DMEM medium for 72 h at 37 °C. The wells were then washed with saline solution, fixed with paraformaldehyde solution (4%; Sigma-Aldrich, St. Louis, MO, USA) and stained with Draq5 (5μM, Biostatus, United Kingdom) to label the cellular DNA. Then, the amount of amastigotes and macrophages in each well was counted in the Operetta High-Content Imaging System (Perkin Elmer, Massachusetts, USA) confocal microscope. Amphotericin B (1 μM; Gibco-BRL, Gaithersburg, MD, USA) was used as a positive control (Chaves et al., 2009). The infectivity index of the parasite was determined by multiplying the mean number of amastigotes per cell by the percentag e of infection (Tanaka et al., 2007). The percentage inhibition of this index was then calculated. P. Anti-T. cruzi activity Murine macrophages adhered in plaque (6x10 3 well -1 ) were infected with T. cruzi Y strain trypomastigotes (6x10 4 trypomastigotes well -1 ) for 24 h. The wells were then washed three times with sterile saline and cells were incubated with the extract and fractions (100 μg mL -1 ) for 72 h at 37 °C under CO2 atmosphere (5%) in DMEM medium. The wells were then washed with saline, fixed with 4% paraformaldehyde solution and stained with Draq5 (4 μM). Quantification of amastigotes and macrophages was performed on the Operetta High-Content Imaging System confocal microscope and the data were expressed as infectivity index. Benzonidazole (5 μM) was used as positive control (Bastos, 2013). This procedure was also approved by Ethics Committee on the Use of Animals (CEUA) of the Gonçalo Moniz Institute /Fiocruz under the number 1126. Q. Statical analisys Data were expressedas the mean ± standard deviation or IC50 values based on three independent experiments. Significant differences (p< 0,05) were detected by oneway ANOVA with Dunnet post-test using GRAPHPAD Prisma (5.0), using significance of 5%. The correlation between antioxidant, trypanocidal and leishmanicidal activity and phenolic content were determined by the Pearson correlation test with significance of 5%.

R. Determination of total phenolic and antioxidant activities
The concentration of phenolics in the extract and fractions of the stem bark of M. rufula is shown in Table 1. The AEFMR fraction had the highest phenolic concentration in the samples (143 μg EAG mg -1 ), whereas HFMR contained about 5 times less phenolic compounds (29 μg EAG mg -1 ).
Extracts and fractions of M. rufula were evaluated by three different antioxidant methods:  DPPH and ABTS + free radical scavenging and inhibition of lipid peroxidation. All samples were able to deactivate  DPPH and ABTS + radicals and inhibit β-carotene oxidation by lipid radicals (Table 1). The MFMR fraction was the most potent in reducing the  DPPH (IC50 = 3 μg mL -1 ) radical compared to the gallic acid standard (IC50 = 1.5 μg mL -1 ). In addition, it was also the most effective fraction in inhibiting the β-carotene decay, with 20 times more potency than trolox standard (IC50 = 21μg mL -1 ). In the case of the ABTS + method, the hexane fraction unexpectedly reduced the radical with IC50 of 0.12 μg mL -1 , but reacted with  DPPH at higher concentrations (IC50 = 60 μg mL -1 ).
In the comparison between the content of reducing compounds, among them phenolics, with the antioxidant activity, the results of the  DPPH and ABTS + assays were not correlated with the concentration of these compounds (r = 0.643 and p = 0.241 and r = 0.645 and p = 0.239, respectively). On the other hand, there was a correlation between these compounds and the β-carotene co-oxidation assay (r = -0.929 and p = 0.022), which suggests that the reducing compounds of the Folin-Ciacalteou reagent are also important in the protection against oxidation of βcarotene.

S. Cell viability, cytoprotection and antioxidant acitivity in cell model
The extract and fractions (50 μg mL -1 ) of the stem bark of M. rufula were effective in protecting macrophagedifferentiated THP-1 against H2O2-induced cell death when pre-incubated for 24 h with the cells before oxidant addition (Fig. 1A). In particular, the ethyl acetate fraction protected the cells in approximately 83%. Furthermore, this fraction inhibited the intracellular oxidation of DCFH mediated by H2O2 in these cells exposed to samples (50 μg mL -1 ) was also greater for the ethyl acetate fraction (Fig.  1B). The activity of an important antioxidant and cytoprotective enzyme, hemeoxygenase, increased almost twice when the cells were pre-incubated with the same fraction (Fig. 1C).
It is important to note that, even under concentrations of up to 100 μg mL -1 of the extract or fraction of M. rufula stem bark, THP-1-derived macrophages remained viable (> 90%).  T. Antibacterial activity Extract and fractions of the stem bark of M. rufula were evaluated for antibacterial potential against gram positive (S. aureus) and negative (P. mirabilis, P. aeruginosa, E. coli and K. pneumoniae) bacteria (Table 2). EEMR and HFMR did not present any antibacterial activity against the bacteria tested at concentrations up to 1 mg mL -1 . EAFMR was active against K. pneumoniae

U. Antiproliferative activity to cancer cells
The antiproliferative activity of the ethanolic extract of M. rufula on HepG2 (human hepatocellular carcinoma) and HL-60 (human promyelocytic leukemia) cancercells was investigated. The extract at the concentration of 50 μg/mL showed weak antiproliferative activity on both cell lines (14.2 and 17.9%, respectively), when compared to doxorubicin (90.3 and 95.0%, respectively).

V. In vitro inhibition of L. amazonensis arginase
Extract and fractions of M. rufula were also evaluated for the ability to inhibit the enzyme arginase of Leishmania amazonensis. The ethanolic extract and the ethyl acetate fraction had the lowest IC50 values (15.7 and 15.6 μg mL -1 , respectively) (Table 3). Quercetin, used as a positive control, inhibited the enzyme with an IC50 of 1.0 μg mL -1 . Furthermore, there were no correlation between phenolic compounds and L. amazonensis arginase inhibition (r = -0.446 and p = 0.451).

W. Anti-L. amazonensis and T. cruzi activities
In macrophages infected with L. amazonensis amastigotes, the ethanolic extract and the hexanic fraction reduced the infectivity index of the parasite to 4.1% and 6.9%, respectively (Table 4). MFMR, HAFMR and EAFMR, however, reduced the infectivity of L. amazonensis amastigotes to 21.4, 24.4 and 19.5%, respectively. Additionally, the extract and fractions of M. rufula were evaluated for in vitro trypanocidal potential. Murine macrophages infected with T. cruzi and treated with EAFMR, MFMR, HAFMR and HFMR had the infectivity index reduced by 78.1, 53.9, 54.9, and 10.5%, respectively (Table 4). There was no association between the effect observed on L. amazonensis amastigotes and the phenolic concentration in the samples (r = 0.862 and p = 0.060). On the other hand, a positive correlation between the concentration of reducing compounds in the samples with the trypanocidal activity was found (r = 0.972 and p = 0.006).

X. Qualitative analysis of phytochemicals
The phytochemical profile of the stem bark of M. rufula was initially analyzed for the presence of triterpenes, alkaloids and flavonoids by some classical phytochemical methods. The assay did not demonstrate the presence of these compounds in the samples, except for HFMR, whose qualitative Liebermann-Burchard test indicated the presence of triterpenes. Recognizing the limitations of these methods (Simões et al., 1999), plant extract and fractions were also analyzed by infrared technique to identify which functional groups were present and to correlate them with groups common to the Sapotaceae family, followed by LC-MS/MS and GC-MS analysis.

Y. Infrared spectroscopy
The IR spectrum (Table 5 and Fig. 2) of the HFMR fraction showed a profile of CH3-and CH2-rich compounds which along with C=O and C-O bands of esters indicate the presence of fatty esters and/or terpenes, common compounds in low polarity fractions of plant extracts. The infrared analysis does not allow the distinction between the groups above mentioned. However, considering the qualitative analysis, it can be stated that HFMR has triterpenes. In the EAFMR fraction, a relative decrease in the intensity of the bands related to fatty esters was observed. In contrast, there was a strong band of O-H groups present in phenols or alcohols, besides the presence of numerous C-O binding bands at 1020 to 1200 cm -1 , indicating the presence of glycosidic groups. On the other hand, the analysis of the spectra allowed to conclude that the chemical profile of the constituents present in the HAFMR fraction was similar to the constituents of the MFMR fraction, being observed an intense band corresponding to O-H groups, which is in agreement with the greater polarity of these fractions, but absence of compounds with C=O groups of esters and ketones. Furthermore, it was found that both fractions had a large number of C-O stretching bands, indicating the presence of glycosidic groups, as well as a band of carbonyl conjugated to aromatic rings, common in classes of compounds such as flavones or aromatic acids (Barbosa, 2013). νC=C (alkene or aromatic ring)  rufula.

Z. LC-MS/MS analysis
The methanolic, hydroalcoholic and ethyl acetate fractions of the M. rufula stem bark were analyzed in the sequence, by negative ionization mass spectrometry. The ions m/z 577 and 865 were found in the EAFMR and HAFMR fractions ( Table 6). The comparison of m/z values and their respective fragments with the literature indicated the presence of proanthocyanidin dimers (Bystrom et al., 2008). Proanthocyanidin trimers (m/z 865.28 and 865.33) were also potentially found in the EAFMR and HAFMR fractions (Simões et al., 1999, Barbosa, 2013. Ions 289 and 577 also suggested the presence of proanthocyanidin subtypes as catechin in ethyl acetate and hydroalcoholic fractions, whereas the 341 ion was possibly the glycoside caffeoyl glucose present in the MFMR fraction (Bastos et al., 2007).
The EAFMR, HAFMR and MFMR fractions presented the ions 757 and 367, whose fragmentation did not allow the identification of the potential compound, but the literature suggests them as quercetin-3-O-triglycoside and feruloylquinic acid (Chen et al., 2012). Besides, the nature of several other ions and their fragments has not been suggested, which demonstrates the importance of further studies in order to reliably isolate and identify these compounds. CG-MS analysis Finally, the HFMR fraction was analyzed by gas chromatography coupled to mass spectrometry, which detected the presence of olean-12-en-3-one, lanosta-8,24dien-3-one, lupenone, lupeol acetate, cycloheucalenol acetate, cyclolanostan-3-ol, cis-3,14-clerodadien-13-ol, by comparison with the NIST library and similarity of 92, 76, 89, 87, 76, 85 and 79%. The chemical structure of the compounds identified in the HFMR is shown in Fig. 3.

IV.
DISCUSSION Species of the genus Manilkara are known to contain flavonoids, phenolic acids, saponins and triterpenes, which are associated with pharmacological properties, including anti-inflammatory, antiparasitic, antitumor, antibacterial and antioxidant properties (Ma et al., 2003;Eibonde et al., 2004;Eskander et al., 2014). Among the different metabolites, phenolic compounds are important due to their inherent antioxidant potential, being able to reduce damage to the host tissue during inflammatory and/or oxidative processes typical of several chronic diseases such as cancer, arthritis, diabetes, atherosclerosis, among others (Fernandes, 2010;Parick and Patel, 2016). Thus, concentrations of phenolics and other reducers in all samples of the stem bark of M. rufula were determined, and the concentrations found here are higher than tho se in the study of Parikh and Patel (2016) from the methanolic extract of the fruit of Manilkara hexandra (8.1 μg EAG/mg) . It is noteworthy that the AEFMR fraction presented higher phenolic concentration in relation to the HFMR fraction likely because most of reducing compounds are more polar (Andreo and Jorge, 2006).
As for the antioxidant activity, it was possible to observe that all the samples were able to deactivate the  DPPH and ABTS + radicals and to inhibit the oxidation of β-carotene by the lipid radicals. Although studies demonstrating the antioxidant activity of M. rufula are missing, investigations with Sideroxylon obtusifolium from same family as M. rufula have shown that the ethanolic extract of this plant reduces the  DPPH radical with IC50 of 9.5 μg/mL, indicating a similar antioxidant activity between these two species (Leite et al., 2015). The fractions of M. rufula probably contain molecules with cytoprotective activity, particularly the ethyl acetate fraction that protected the cells against death induced by H2O2 in approximately 83%. This same fraction also inhibited intracellular redox stress more significantly than tempol, a well recognized antioxidant in several in vitro and in vivo models (Soule et al., 2007). The ethyl acetate fraction may protect the macrophages against H2O2induced damage through Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway, which is an important transcription factor for many antioxidant and cytoprotective enzymes, including hemeoxygenase. A recent study with Madhuca indica from Sapotaceae family showed that the compound 3,5,7,3',4'-pentahydroxyflavone was cytoprotective by inducing the expression of antioxidant enzymes related to Nrf2-Keap1 system (Wang et al., 2010). Indeed, Nrf2 signaling pathway may be activated by several phytochemicals, including polyphenols and triterpenoids , which could explain the effects mediated by M. rufula on macrophages (Park et al., 2007;Facundo et al., 2005). An intriguing point is that all the samples of M. rufula showed considerable toxicity against THP-1-derived macrophages up to the concentration of 100 μg/mL. In the literature, the ethyl acetate fraction of Pouteria venosa (Sapotaceae) plant showed no toxicity on macrophages of the J774 lineage at the concentration of 200 μg/mL, which is in agreement with our results (Santos et al., 2015). Therefore, these preliminary data suggest that this plant possibly has low toxicity for application in in vivo models.  Scientific and ethnopharmacological studies have also reported the use of Sapotaceae plants in the treatment of cancer (Bhaumik et al., 2015). Nevertheless, the EEMR showed only weak antiproliferative activity on HepG2 and HL-60 cell lines. The ability of some constuints from this plant in activating the Nrf2 pathway may justify, at least in part, its low toxicity against cancer cells (Chen et al., 2015).
Sapotaceae also contains plant species to which antiprotozoal activity has been reported, including against Trypanosoma, Plasmodium and Trichomonas vaginalis In the case of macrophages infected with L. amazonensis amastigotes, the MFMR, HAFMR and EAFMR fractions reduced the infectivity of L. amazonensis amastigotes more sharply, which leads us to infer that these fractions act on other targets associated with the survival of L. amazonensis (Singh et al., 2012). In contrast, it was observed that EEMR inhibited arginase in vitro, but did not reduce the infectivity of Leishmania in a cellular model, which could be due to lower permeability of phytochemicals through the membranes or to metabolism of these compounds by the parasite/macrophage . Moreover, there was no association between the effect observed on L. amazonensis amastigotes and the phenolic concentration in the samples, which indicates that other compounds must be responsible for the leishmanicidal activity. The IC50 of arginase inhibition by fraction EAFMR, MFMR, HAFMR are closed related to the reduction of infectivity index of L. amazonensis amastigotes showed in this study. These data are an evidence of the extract and fractions kill L. amazonensis targeting parasite arginase. Nevertheless, the development of therapeutic strategies based on antioxidants sources should take into account the potential risk of altering host resistance to parasite infection and worsering the infection (Silva and Castilhos, 2015).
Regarding the in vitro trypanocidal activity, all fractions significantly reduced T. cruzi infection (10.5 to 78.1%). There are no specific studies on the leishmanicidal and trypanocidal activities of M. rufula in the literature. However, a study demonstrated the trichomonicidal effect of the dichloromethane extract of leaves and branches of this plant . The aqueous extract from the leaves of P. ramiflora (Sapotaceae) was able to induce the death of promastigote forms of Leishmania amazonensisin vitro (Linares et al., 2008). Cruz et al. (2010) found that a flavonoid glycoside present in leafs of Cecropia pachystachya (Cecropiaceae) altered the mitochondrial DNA of L. amazonensis, besides inhibiting the enzyme arginase, and thus preventing the development of this parasite. In addition, the triterpenoids isolated from the dichloromethane extract of the fruit pericarp of Omphalocarpum procerum (Sapotaceae) led to the death of T. cruzi, L. donovani, P. falciparum and T. brucei rhodesiense in different cell models (Ngamgweet al., 2014).
The present study indicated the presence of several types of terpenes, such as proanthocyanidins , in M. rufula from Maracás, Bahia, Brazil. Previous studies with M. zapota and M. rufula had isolated several types of proanthocyanidins, which is in agreement with the present findings (Wang et al., 2010). Based on these preliminary results, the pharmacological activities described for M. rufula can be explained by the presence of proanthocyanidins, as they are widely known in the literature because of their biological activities such as anti-inflammatory, anticancer, antibacterial, antifungal, antiviral, antiparasitic activity, inhibition of platelet aggregation and cytoprotective (Augustin et al., 2011;Trentin et al., 2013). However, this can only be affirmed after isolation of M. rufula metabolites for reliable identification of plant components.