Activity of alkaloids from Aspidosperma nitidum against Leishmania (Leishmania) amazonensis

This study evaluated the morphological changes caused by fractions and subfractions, obtained from barks of Aspidosperna nitidum, against L. (L.) amazonensis promastigotes. The ethanolic extract (EE) obtained through the maceration of trunk barks was subjected to an acid–base partition, resulting the neutral (FN) and the alkaloid (FA) fractions, and fractionation under reflux, yielded hexane (FrHEX), dichloromethane (FrDCL), ethyl acetate (FrACoET), and methanol (FrMEOH) fractions. The FA was fractionated and three subfractions (SF5-6, SF8, and SF9) were obtained and analyzed by HPLC–DAD and 1H NMR. The antipromastigote activity of all samples was evaluated by MTT, after that, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for the active fractions were performed. Chromatographic analyzes suggest the presence of alkaloids in EE, FN, FA, and FrDCL. The fractionation of FA led to the isolation of the indole alkaloid dihydrocorynantheol (SF8 fractions). The SF5-6, dihydrocorynantheol and SF-9 samples were active against promastigotes, while FrDCL was moderately active. The SEM analysis revealed cell rounding and changes in the flagellum of the parasites. In the TEM analysis, the treated promastigotes showed changes in flagellar pocket and kinetoplast, and presence of lipid inclusions. These results suggest that alkaloids isolated from A. nitidum are promising as leishmanicidal.


Results
Phytochemical studies. The yields of EE and fractions are described in Table 1 The analysis of the 1 H NMR spectrum of the SF5-6 subfraction, showed signs of hydrogen with displacements in δH 7.47, 7.35, 7.07, and 7.19 that correspond to aromatic hydrogens. Meanwhile, SF8 1 H NMR spectrum (see signs below) demonstrated the presence of three signals with chemical shifts in δH 7.40 for H9, δH 7.28 for H12, Table 1. Yields of ethanolic extract and its fractions obtained by acid-base partition or reflux extraction. a EE:ethanolic extract; b FA: alkaloids fraction; c FN: neutral fraction; d FrHEX: hexane fraction; e FrDCL: dichloromethane fraction; f FrACOET-ethyl acetate fraction; g FrMEOH-methanolic fraction.   Anti-promastigote activity and morphological analysis. When considering the antipromastigote activity, Table 2 shows the 50% inhibitory concentrations (IC 50 %) and that only samples SF5-6, SF-9 and dihydrocorynantheol were active, while FrDCL was moderately active.
In the scanning electron microscopy (SEM) analysis, promastigotes treated with the dichloromethane fraction of EE (FrDCL) displayed alterations in a concentration-dependent fashion. We observed cell rounding, membrane septation and shortening of the flagellum size in parasites treated with 50 µg/mL of FrDCL (Fig. 3F). At a concentration of 25 µg/mL, promastigotes still presented rounded-shape cells (Fig. 3G), and in parasites treated with 12.5 µg/mL, there were no significant changes (Fig. 3H).
Similarly, in parasites treated with SF5-6, SF8, and SF9, as analyzed by SEM, changes were concentrationdependent. For the parasites treated with 50 µg/mL of SF5-6, a short flagellum was observed in most of them, as well as a double flagellum and small protuberances along the flagellum in some of the promastigotes (Fig. 3I), and cell rounding. At the lowest concentration (25 µg/mL), the features displayed were flagellum shortening and cell rounding (Fig. 3J).
The parasites treated with 25 µg/mL of dihydrocorynantheol presented a change in shape of the cell body, which was septate, and flagellum shortening (Fig. 3K). Promastigotes treated with 12.5 µg/mL displayed rounded shape, some displaying cell body septation, but no promastigotes with a short flagellum were observed (Fig. 3L). For the parasites treated with 6.25 µg/mL of dihydrocorynantheol, only rounding cells were observed (Fig. 3M).
Moreover, for the parasites treated with 50 µg/mL of SF9, a round shape and protuberances were observed along the flagellum (Fig. 3N), while at 25 µg/mL there were cells with rounded shape (Fig. 3O), and at 12.5 µg/ mL most parasites presented an elongated cell body, a long flagellum, and no protuberances (Fig. 3P).
No significant changes were observed for the negative control group (NC; Fig. 3A), or for the solvent control group (CSOL; Fig. 3B). The main changes caused by the treatment with Amphotericin B were changes in flagellum size, loss of elongated shape, septation of the cell body (0.1953 µg/mL; Fig. 3C). For the promastigotes treated with 0.09765 µg/mL, the same changes were observed, except for the changes in flagellum size (Fig. 3D). The promastigotes treated with 0.048825 µg/mL presented more elongated cells, but septation or flagellum shortening was not observed (Fig. 3E).
Additionally, other groups of promastigotes were treated with the same samples (FrDCL, SF5-6, SF8, and SF9) and the morphological changes were evaluated by transmission electron microscopy (TEM). For the NC, no subcellular changes were observed (Fig. 4A), as well as in the CSOL (Fig. 4B). Parasites treated with FrDCL (50 µg/mL) presented important cellular alterations, such as complete loss of normal morphology, retraction of the cell body, cytoplasmic disorganization, dilated flagellar pocket with evagination of its membrane for possible    www.nature.com/scientificreports/ formation of vesicles, and cytoplasmic vacuolization (Fig. 4C). The following changes were observed for the parasites treated with 25 µg/mL of FrDCL: rounding cells, lipid accumulation, apparently fragmented chromatin in the nucleus, and an increase in the number of vacuoles in the cytoplasm (Fig. 4D). The treatment of promastigotes with 12.5 µg/mL induced dilatation of the flagellar pocket and alteration in the flagellar membrane (Fig. 4E).
In promastigotes treated with 50 µg/mL of SF5-6, an enlarged flagellar pocket with evagination of its membrane for possible formation of vesicles and alteration of the flagellar membrane were observed (Fig. 4F).
Treatment of promastigotes with 25 µg/mL of dihydrocorynantheol induced cytoplasmic disorganization, cell body retraction, complete loss of normal flagellar pocket morphology with vesicles which were probably results of the evagination of the flagellar pocket membrane, and presence of material with a granular aspect inside these vesicles. In addition to these changes, lipid inclusions and vacuoles were observed in the cytoplasm (Fig. 4G). The treatment of promastigotes with 12.5 µg/mL promoted the appearance of lipid inclusions, changes in the flagellar pocket membrane, which presented vesicles with granular material and were probably the result of this evagination, and dilation of cisterns of the Golgi complex (Fig. 4H). For the promastigotes treated with 6.25 µg/mL, there was also a change in the shape of the flagellar pocket and vesicles within presented granular material (Fig. 4I).
For the promastigotes treated with SF9 at 50 µg/mL, we observed retraction of the cell body, alteration in the flagellar pocket morphology, which contained vesicles with granular material inside, kinetoplast dilation, and the presence of small vacuoles in the cytoplasm (Fig. 4J). Promastigotes treated with 25 µg/mL displayed changes in the flagellar pocket shape, dilated kinetoplast, presence of lipid inclusions, and small projections of the parasite's cytoplasmic membrane (Fig. 4K).

Discussion
To date, no studies that tested the antileishmanial activity of dihydrocorynantheol were found, and this study is the first to test the activity of this indole alkaloid against the parasite of the genus Leishmania. Notwitstanding, previous studies isolated different compounds from A. nitidum: 10-methoxy-dihydrocorynantheol, corynantheol 13 , harman carboxylic acid 14 , 3-methyl-harman-carboxylic acid, dihydrocorynantheol, dehydrositsiriquine 15 , and braznitidumine 16 . However, none of these alkaloids isolated from A. nitidum, and which have a structure similar to dihydrocorynantheol, were tested against species of the genus Leishmania, and only one close study was identified, in which the author tested the alkaloid braznitidumine, isolated from the methanolic extract of A. nitidum, against the protozoan Plasmodium falciparum, but is was inactive 17 . In the present study, dihydrocorynantheol (SF8) was isolated, identified, and tested against L. (L.) amazonensis, and preliminary analyzes suggest that the other fractions (SF5-6 and SF9) are indole alkaloids, but it was not possible to determine their structure.
After analysis in HPLC-DAD, we believed that the samples where alkaloids and would be promising as leishmanicidal agents, and that fractionation would contribute to this activity. However, only the subfractions (SF5-6; dihydrocorynantheol, and SF-9), which were suggestive of alkaloids, displayed activity against L. (L.) amazonensis, whereas the FA was inactive. These results suggest alkaloids isolation is important to antipromastigote activity. Indeed, many alkaloids isolated from plants showed leishmanicidal properties 18 .
Some studies describe natural compounds as capable of promoting morphological changes in parasites 19,20 . The alkaloid β-carboline-1-propionic acid, can induce apoptosis 21 and it can induce great ultrastructural damage in L. amazonensis promastigotes, including some evidence of apoptosis, such as vacuolization of the cytoplasm, presence of myelin-like figures, and swollen kDNA networks 22 . Another alkaloid, voacamine, induced intense cytoplasm disorganization, presence of autophagic vacuoles, changes in the kinetoplast, mitochondrial membrane, and mitochondrial ridges in promastigotes 19 .
Ultrastructural analysis (SEM and TEM) of promastigote forms treated with FrDCL, SF5-6, SF8, and SF9, displayed great morphological changes, which occurred in a concentration-dependent fashion. Despite the fact that FrDCL presented a moderate antipromastigote effect, based on studies developed by members of this research group, and due to the presence of metabolites other than alkaloids in this fraction, we decided to include it in this study to check whether the combination of molecules could promote a different set of morphological changes upon the parasites, than the isolated alkaloids.
In this sense, the morphological analysis revealed that parasites treated with FrDCL presented changes in shape, in the flagellar pocket, and in the cytoplasm. The reduction in concentration contributed to damage reduction, causing dilation of the flagellar pocket and alteration in the flagellar membrane. The FrDCL fraction externalization by the parasite through vesicles resulting from the evagination of the flagellar pocket membrane, suggests that this sample, perhaps, could inhibits hydrolytic 23,24 or proteolytic enzymes 25 and thus be responsible for the damage to the parasite. However, further investigations are needed to elucidate the possible mechanism of action.
Studies reported that endocytosed material is internalized from the flagellar pocket, and then vesicles sprout out from the pocket membrane. These vesicles appear to fuse and discharge their content into intracellular organelles comparable to the endosomes of mammalian cells 23,24 . There is little information about the factors that control access to the flagellar pocket, and about the physical and biochemical properties of constituents of the pocket lumen. The presence of certain hydrolases (eg, acid phosphatase) within the lumen of the flagellar pocket is an indication that the pocket may act as a prelysosomal hydrolytic compartment 23,24 .
When considering the study about microorganism proteases, it is important to keep in mind that these enzymes play important roles in the physiology of these organisms, as well as in the pathologies caused by them 26 . Among them, cysteine proteases are the most investigated proteolytic enzymes in Leishmania. These enzymes play important roles in Leishmania, such as virulence, viability maintenance, parasite morphology, invasion of the host's mononuclear phagocytic system, and the modulation of its immune response 27,28 , thus constituting attractive targets for chemotherapy for the treatment of leishmaniasis. www.nature.com/scientificreports/ Studies carried out the purification and biochemical characterization of three serine proteases from Leishmania (L.) amazonensis promastigotes called LSP 29 , restricted mainly to intracellular compartments 30 , as megasomes and to the flagellar pocket 31 .
Protease activity can be regulated in cells or organisms in different ways, including protease inhibition through specific inhibitors 32 . These inhibitors are valuable tools for investigating the biochemical properties and biological functions of proteases 33 and the inhibition of these enzymes by specific inhibitors can be an important strategy in the production of potent antimicrobial agents 34 .
When analyzed by TEM, the L. (L.) amazonensis promastigotes treated with 10−5 M of the protease inhibitor ShPI-I, obtained from the sea anemone Stichodactyla helianthus, presented flagellar pocket with vesicles inside, in addition to bubble formations in the membrane of its pocket, a structure of intense exocytic/endocytic activities. In the cytoplasm, the presence of vesicles that resemble autophagic vacuoles was observed, and this would a be result of intense exocytic/endocytic activity induced by this inhibitor. Moreover, all parasites exhibited changes in shape 25 . These changes were not seen in control cells. Thus, other classic protease inhibitors such as N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and benzamidine (Bza) were tested in this study and they also caused changes in the flagellar pocket. In another study, it was observed that ultrastructural abnormalities in the flagellar pocket and lysosome/endosome were seen exclusively with cysteine protease inhibitors regardless of their chemical composition, for example vinyl sulfone versus dihydrazide. This observation suggests that the cellular alterations seen are due specifically to inhibition of the cysteine proteases of Leishmania 27 .
We highlight that changes in the flagellar pocket and parasites shape were also observed in the present study. However, whether these enzymes participate in the exocytosis/endocytosis pathway through the processing of intracellular proteins or even in the morphological maintenance of Leishmania remains to be elucidated 25 .
Similar to FrDCL, treatment with a lower concentration of SF5-6 promoted changes in the shape of the flagellar pocket, which presented vesicles with granular material. Increasing the concentration of treatments increased the intensity and occurrence of damage.
The promastigotes treated with FrDCL and SF5-6 presented lipid inclusions. The biogenesis of lipid bodies is involved in cellular homeostasis in eukaryotes, as well as during infection by intracellular pathogens 35 . Given the role in cellular homeostasis, the accumulation of lipid bodies observed in the treated parasites may be a result of cellular disturbances and loss of homeostasis. Moreover, the intracellular presence of lipid bodies can indicate changes in the content of phospholipids and sterols 36 .
Similarly, changes in flagellar pocket were observed in parasites treated with dihydrocorynantheol. In addition, the retraction of the cell body of the parasites was observed, which is an alteration frequently detected in apoptotic cells 37 . During the initial stages of apoptosis, rounding and retraction of the cell is observed. This occurs due to constituent's proteolysis of the cytoskeleton 38 , facts that were observed in the present study.
Among other changes caused by dihydrocorynantheol, there is the dilation of cisterns in the Golgi complex. This organelle is essential for the processing of lipids and proteins from the endoplasmic reticulum (ER) 39 . Like the endoplasmic reticulum, the Golgi complex can trigger the apoptotic process in response to stressful situations (excess of proteins unfolded from the endoplasmic reticulum, changes in membrane traffic or in its structure, among others). Some studies demonstrate that the Golgi complex not only suffers the consequences of the apoptotic process, but also plays an active role in the triggering of this type of cell death 40 . All of this reinforces the hypothesis that the leishmanicidal mechanism of dihydrocorynantheol should involve apoptosis.
In the treatment of promastigotes with the SF9, changes in shape, flagellar pocket, kinetoplast, presence of vacuoles, lipid inclusions and small projections of the parasite's cytoplasmic membrane were observed. Most changes are similar to the ones induced by other alkaloids and FrDCL. It is worth mentioning that treatment with SF9 induced the alteration in the kinetoplast of the parasites. This change can be a result of the fragility of the surrounding mitochondrial membrane. Thus, changes in the mitochondrial membrane could indirectly explain the changes observed in the kinetoplast 41 . The kinetoplast appears physically associated with the mitochondrial membrane and the basal body by thin filaments, which forms a complex structure known as the tripartite binding zone, which is essential for the positioning of the mitochondrial genome and its correct segregation during cell division 42 .

Conclusion
In this study, alkaloids isolated from A. nitidum are promising as leishmanicidal and changes in flagellar pocket may be involved in this activity. Among alkaloids, the most promising is dihydrocorynantheol and, perhaps, apoptosis is involved in this activity. However, further studies need to be carried out to confirm the occurrence of this process in these parasites, since specific markers for apoptosis were not used in this investigation.

Methods
Plant material. The bark of A. nitidum, locally known as Caranapauba were collected in Santa Bárbara do Pará, PA-Brazil (S 01° 10 ′946' W 048° 11 ′715') during May 2013. The plant material was identified by Dra. Rafaela Trindade and the exsiccate was incorporated into the MG Herbarium of the Museu Paraense Emílio Goeldi, under no. MG206608. In the present study we used a wild plant collected from a virgin forest of the Amazon, and our work posed no risk of extinction for the species. During the collection, we took all care to remove the barks so as not to cause any damage to the species, in addition, only a small proportion of the barks were collected. The species were kept integrated and survived the collection. The project complies with national and international guidelines and legislation and is registered on the platform of the national management and Genetic Heritage System and Associated Traditional Knowledge (SISGEN), whose provided license to collect the species under registration A92C186. 2), grounded, and subjected to maceration (7 days) to obtain the EE, which was subjected to an acid-base partition, resulting in the neutral (FN) and alkaloid fractions (FA) 43 . The EE was also subjected to extraction under reflux, yielding the following fractions: hexane (FrHEX), dichloromethane (FrDCL), ethyl acetate (FrAcOEt) and methanol (FrMeOH) 44 .
The analysis was performed in a RP 18 column (45 × 250 mm, 5 µm), using a mixture of water acidified with 0.01% of trifluoroacetic acid (eluent A; Tédia) and acetonitrile acidified with 0.01% of trifluoroacetic acid (eluent B; Tédia) as the mobile phase. A linear elution gradient was used: 70-30% of eluent B for 15 min, 60-40% of eluent B for 20 min and 50-100% of eluent B for 25 min. The temperature was maintained above 26 °C, flow of 1 mL/min, UV detection at 220-400 nm 43 .
Antipromastigote activity. The promastigote form of Leishmania (L.) amazonensis (MHOM/BR/2009/ M26361) was grown in NNN medium (Novy-Nicolle-Mcneal) and later transferred to RPMI (Roswell Park Memorial Institute medium; Gibco) supplemented with 10% of denatured fetal bovine serum (Cultilab materials for cell culture), penicillin G 10,000 IU (Gibco)/mL and streptomycin 10,000 µg (Gibco)/mL and maintained at 25° C ± 1 °C through weekly passages. In the assay of antipromastigote activity, promastigotes were quantified in a Neubauer chamber (Improved) and then distributed in cell culture plates (Techno Plastic Products-TPP), previously pre-dosed with different concentrations of the samples and amphotericin B (positive control; Cristália pharmaceutical products). The plates were incubated and after 72 h of treatment, the tetrazolium salt (Sigma-Aldrich) was added followed by a new incubation. After 4 h of incubation, dimethyl sulfoxide (Dinâmica) was added and, then, the reading was performed in a microplate reader (Biotek, model ELX 808) at 490 nm. The samples were considered active when the IC 50 ≤ 100 µg/mL, moderately active IC 50 101-200 µg/mL, inactive when the IC 50 ≥ 200 µg/mL as adapted from 45 . The 50% Inhibitory Concentration (IC 50 ) is the concentration that causes a 50% reduction in growing cells (viable) and was determined by the GraphPad Prism software.
Morphological analysis. The parasites treated with the active samples were analyzed by SEM (LEO 1450 VP) and TEM (ZEISS model EM 906 e ZEISS model EM 900), according to the method described by 46 .
In the TEM analysis, the parasites were centrifuged, and the pellet resuspended in a 1% glutaraldehyde solution with 4% paraformaldehyde in 0.1 M PHEM buffer (pH 7.4), and 2.5% sucrose for fixation for 1 h. After fixation, the cells were washed with 0.1 M sodium cacodylate buffer (pH 7.4) and post-fixed in a solution containing 1% osmium tetroxide and 1% potassium ferrocyanide for 1 h in the dark, washed with buffer 0.1 M cacodylate (pH 7.4), and then with distilled water and immersed in a contrasting 1% uranyl acetate (Electron Microscopy Sciences) solution in 25% acetone (MERCK) for 1 h. Then, samples were dehydrated in graded acetone (50, 70, 90 and 100%). Afterwards, the cells were slowly impregnated in increasing concentrations of Epon (Electron Microscopy Sciences) diluted in acetone until pure Epon (100%). The samples were then infiltrated with pure Epon + DMP-30 (2,4,6-Tris (dimethylaminomethyl) phenol) and left in a support for polymerization in an oven at 60 °C for 48 h. The blocks were cut in an ultramicrotome (Leica, model EMVC6) and ultrathin sections were contrasted with 5% uranyl acetate and, subsequently, with lead citrate for further analysis by TEM (ZEISS model EM 906 and ZEISS model EM 900).