4-Acetoxydolastane Diterpene from the Brazilian Brown Alga Canistrocarpus cervicornis as Antileishmanial Agent

Natural marine products have shown an interesting array of diverse and novel chemical structures with potent biological activities. Our study reports the antiproliferative assays of crude extracts, fraction and pure compound (4R,9S,14S)-4α-acetoxy-9β,14α-dihydroxydolast-1(15),7-diene (1) obtained from brown alga Canistrocarpus cervicornis showing the antileishmanial activity. We showed that 1 had a dose-dependent activity during 72 h of treatment, exhibiting IC50 of 2.0 μg/mL, 12.0 μg/mL, and 4.0 μg/mL for promastigote, axenic amastigote and intracellular amastigote forms of Leishmania amazonensis, respectively. A cytotoxicity assay showed that the action of the isolated compound 1 was 93.0 times less toxic to the macrophage than to the protozoan. Additionally, compound 1 induced ultrastructural changes, including extensive mitochondrial damage; decrease in Rh123 fluorescence, suggesting interference with the mitochondrial membrane potential; and lipid peroxidation in parasite cells. The use of 1 from C. cervicornis against L. amazonensis parasites might be of great interest as a future alternative to the development of new antileishmanial drugs.

countries, in combating diseases [6]. This is particularly true for marine natural products, which show an interesting array of diverse and novel chemical structures with potent biological activities [28].
Our study reports the antiproliferative assays of crude extracts (EACE, MCE and DCE), fraction (EAF) and pure compound (1) (4R,9S,14S)-4α-acetoxy-9β,14α-dihydroxydolast-1(15),7-diene ( Figure 1) obtained from brown alga C. cervicornis showing the dose-dependent effect against promastigote forms of L. amazonensis (Figures 2 and 3). In Table 1, we have demonstrated the concentrations values of crude extracts, fraction, and 1 that inhibited 50% of this parasite (IC 50 values) after 72 h of incubation. Thus, the IC 50 of crude extracts EACE, MCE and DCE were 50.0 µg/mL, 100.0 µg/mL and 20.0 µg/mL, respectively. The IC 50 of fraction (EAF) and 1 were 8.0 µg/mL and 2.0 µg/mL, respectively. The student's t test (p < 0.05) indicated significant differences between crude extracts, fraction and isolated compounds compared to the control group. In addition, amphotericin B showed IC 50 of 0.06 µg/mL against promastigote forms after 72 h of treatment.    The cytotoxicity on macrophage strain J774G8 of crude extracts, fraction, and isolated compound were also evaluated (Table 1). When macrophages were treated with crude extracts EACE, MCE, and DCE the 50% cytotoxic concentration (CC 50 ) were 50.0 µg/mL, 51.0 µg/mL, and 46.0 µg/mL, respectively. The CC 50 of fraction (EAF) was 47.0 µg/mL and CC 50 of 1 was 186.0 µg/mL. The cytotoxicity to the macrophage and the activity against the protozoan were compared by using the selectivity index (SI). The better results of SI for promastigote forms were obtained with fraction EAF (SI = 5.88) and isolated compound 1 (SI = 93.0). So, EAF and 1 were respectively 5.88 and 93.0 times less toxic to the macrophage than to the protozoan.
The pure compound 1 was more active than crude extracts and fraction. Thus, further experiments were carried out using only this compound. Isolated compound 1 had the activity investigated against axenic amastigote forms of L. amazonensis ( Figure 3). The IC 50 obtained value was 12.0 µg/mL. The effect of the 1 on intracellular amastigotes was observed during 24 h of incubation ( Figure 4). Treatment of intracellular amastigotes with 5.0, 15.0, and 30.0 µg/mL with 1 resulted in decreases in the survival index of 56.0, 72.0, and 76.0%, respectively, compared to the control. Additionally, the IC 50 was 4.0 µg/mL. These results were significant at p ≤ 0.05 as compared to the control group, by the student's t test. In this regard, the result of this preliminary study is very encouraging because it is the amastigote forms of Leishmania that persist in the human host and directly cause all of the clinical manifestations of leishmaniasis [29]. Amphotericin B showed IC 50 of 0.25 and 0.35 µg/mL against axenic amastigote and intracellular amastigote forms, respectively. To find cellular targets in leishmania cells treated with 1, TEM and flow cytometry techniques were employed. We verified that 1 produced ultrastructural alterations in L. amazonensis. Figure 5 shows transmission electron microscopy photomicrographs of promastigotes treated with the isolated compound 1. Figure 5A shows untreated cells with a terminal flagellum, nucleus, and the single mitochondrion with a branched structure, characteristic of this group of organisms, containing a disk-shaped aggregate of DNA called a kinetoplast. In L. amazonensis treated with 1, we highlight that the main alteration which occurred in the mitochondria of the parasite, was seen as intense mitochondrial swelling, as seen in Figure 5B-F.
In accordance with these findings, tests using specific markers for mitochondria were performed. Data obtained from a flow cytometry by using Rh123 presented in Figure 6 showed a marked decrease in the percentage population of upper right gate (76.55% and 68.71%). This indicates depolarization of the mitochondrial membrane potential in the cells following treatment with 1 at 50 µg/mL and 100 µg/mL, respectively ( Figure 6). Similarly, a decrease in membrane potentials was also observed following treatment with the standard drug Carbonyl Cyanide m-chlorophenylhydrazone (CCCP) (75.37%) at 200 µM for 3 h at 32 °C. In contrast, untreated cells maintained the membrane potential (98.41%) ( Figure 6, upper right quadrant). The student's t test (p < 0.05) indicated significant differences between cells treated with copaiba oil compared to the negative control group.  Mitochondrion found in trypanosomatid parasites had distinct structural and functional characteristics of mammalian cells making this organelle an exceptionally attractive chemotherapeutic target. Likewise, investigations have shown that mitochondria can be targeted by different drugs for Leishmania sp. [13,14,[30][31][32]. Additionally, several studies have reported that mitochondrial alterations may lead to programmed cell death by apoptosis [33][34][35][36].
In order to evaluate the effects of the compound 1, the lipid peroxidation of cell membrane was determined. It was assessed by measuring TBARS in leishmanial cells after treatment with 1 compared to control or untreated cells (Figure 7). In addition, thenoyltrifluoroacetone (TTFA), a conventional mitochondrial complex II inhibitor, was used as positive control. Compound 1 treatment at 100 µg/mL displayed an increase in lipid peroxides after 6 h of drug treatment, with a 3.4-fold lipoperoxidation compared to the untreated control cells while the increase in lipid peroxidation obtained with the TTFA was 2.1-fold compared to the control. Previous studies have demonstrated that loss of the mitochondrial membrane potentially induces formation of reactive oxygen species (ROS) inside cells, which induces lipid peroxidation. Moreover, the cellular ROS generation in turn leads to the damage of the oxidative citoplasmatic membrane, and DNA lesions [33,34,37,38].

Figure 7.
Effect of compound 1 on lipid peroxidation (production of malondialdehyde (MDA)) of amastigote forms of Leishmania amazonensis. Each bar represents the mean ± standard error of at least three independent experiments, which were performed in duplicate. * Significant difference of each group from the control (p < 0.05).

Algal Collection
Specimens of the brown macroalga C. cervicornis (Dictyotaceae, Phaeophyta) were collected by free diving off the coast of Paraíso Beach, Pernambuco State, Brazil (08°21′S, 34°57′W), in October 2009, at depths between 1 and 2 m. The algal material was cleaned manually from epiphytic organisms and air-dried immediately after collection. Voucher specimens were deposited in the Herbarium of Universidade Federal de Pernambuco, Recife, Brazil (62948).

Chemical Extraction
The air-dried algal material (300.0 g of C. cervicornis) was extracted in dichloromethane (CH 2 Cl 2 ) at room temperature for 30 days and the solvent was evaporated in vacuo by rota-evaporation, yielding 14.6 g of dry dichloromethane crude extract (DCE). The residue was further extracted in ethyl acetate (EtOAc) using similar procedures, yielding 0.9 g of dry ethyl acetate crude extract (EACE), and finally extracted in methanol (MeOH), yielding 0.3 g of dry methanol crude extract (MCE).
The fractions F26-F38 eluted with EtOAc from DCE showed similar results when analyzed by TLC, and then they were combined to produce a brownish residue (1.3 g) which was named EAF (ethyl acetate fraction).
All the crude extracts formed (EACE, DCE, and MCE), fraction (EAF) and pure compound isolated 1 were monitored by TLC using silica gel GF 254 (Merck) as stationary phase and n-hexane/EtOAc 3:1 and 4:1 as mobile phase. The chromatoplates used were revealed through spraying it with a solution of ceric sulphate and sulfuric acid acquired by 2.1 g of Ce 2 (SO 4 ) 3 .4H 2 O; 21 mL of H 2 SO 4 and 300 mL of H 2 O, followed by heating at 100 °C for 3 min.

Parasites
Leishmania promastigotes were grown in Warren's medium (brain-heart infusion plus hemin and folic acid) pH 7.2, supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco Invitrogen Corporation, New York, USA), at 24 °C in a tissue flack. The strain used was Leishmania amazonensis (MHOM/BR/Josefa) originally isolated from a human case of difuse cutaneous leishmaniasis.
Leishmania axenic amastigotes were obtained by in vitro transformation of infective promastigotes [40]. These forms were maintained in Schneider's medium (Sigma, St. Louis, MO, USA) pH 4.6, supplemented with 20% heat-inactivated FBS, at 32 °C in a tissue flask.

Antileishmania Activity in Vitro against Promastigotes and Axenic Amastigotes
The growth inhibition test that was performed on promastigotes forms of L. amazonensis from a 48 h logarithmic phase culture was suspended to yield 1 × 10 6 parasites/mL. It was then cultivated in 24-well culture plates at 25 °C in Warren's medium, supplemented with 10% FBS in the presence or absence of increasing concentration of 1 for 72 h. In a same way, axenic amastigotes forms of L. amazonensis from 72-h-old logarithmic-phase culture were suspended to yield 1 × 10 6 parasites/mL, and then were cultivated in 12-well culture plates at 32 °C in Schneider's medium. Next they were supplemented with 20% FBS in the presence or absence of increasing concentration of compound 1 for 72 h. Amphotericin was used as a positive control. DMSO was used for the solubilized drugs, but the final DMSO concentration did not exceed 0.5%, which did not show deleterious effects on the parasites. The leishmanicidal activity was determined by direct counting of the cells in a Neubauer chamber and the 50% inhibition concentration (IC 50 ) was obtained graphically by plotting concentration versus percentage of growth inhibition.

Activity against Intracellular Amastigotes
For assay of the effects of the compound on intracellular amastigotes, peritoneal macrophages of BALB/C, mice were used. Peritoneal macrophages (5 × 10 5 cells/mL) were plated on coverslips (diameter 13 mm) in 24 well plate in RPMI 1640 medium supplemented with 10% inactive FBS, and incubated for 24 h at 37 °C in a 5% CO 2 atmosphere for adherence, after the peritoneal macrophages were infected with promastigotes of L. amazonensis in multiples of 10 parasites per host cell and incubated at 37 °C in a 5% CO 2 atmosphere. After 6 h of infection, infected macrophages were treated with compound in concentrations 10, 20 and 30 µg/mL and incubated 24 h again. Afterwards the monolayer's were washed with PBS, fixed with methanol, and stained with Giemsa. The percentage of infected macrophages and the mean numbers of amastigotes/infected macrophage were determined by counting at least 200 macrophages in duplicate cultures, and results were expressed as shown in the survival index. The survival index was obtained by multiplying the percentage of macrophages cells with parasites by the mean number of internalized parasites per cell.

Cytotoxicity Assay
The cytotoxicity assay was performed in 96-well plates. A suspension of 5 × 10 5 J774G8 cells in RPMI 1640 medium supplemented with 10% FBS was added to each well in 96-well microtiter plates.
The plates were incubated in a 5% CO 2 -air mixture at 37 °C to obtain confluent growth of the cells. After 24 h, the medium was removed and the cells were treated with several concentrations of compounds and the plates were incubated for 48 h. Control cells without compound were included. The adhered macrophages were fixed with 50 μL/well of 10% trichloroacetic acid at 4 °C for 1 h; after that, the well plates were washed with water, and attained with 50 μL/well of sulforhodamine B (0.4% w/v) in 1% acetic acid solution; the microplate was then maintained at 4 °C for 30 min. Next, the microplate was washed five times with 1% acetic acid to remove the sulforhodamine B, then 150 μL/well of 10 mM unbuffered Tris-base solution (Sigma) was added. The absorbance of each individual well was read at 530 nm. Dose-response curves were plotted (values expressed as percentage of control optical density) and the 50% cytotoxicity concentration (CC 50 ) was determined by logarithm regression analysis of the data obtained.

Transmission Electron Microscopy
Ultrastructural analysis was performed on promastigote forms of L. amazonensis treated with 2.0 and 10.0 µg/mL of the compound 1, after 48 h for treatment, the parasites were washed in 0.1 M phosphate-buffered saline and fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4 °C. The cells were post fixed in a solution containing 1% osmium tetroxide, 0.8% potassium ferrocyanide, and 5 mM calcium chloride in 0.1 M cacodylate buffer. Next they were washed in the same buffer, dehydrated in increasing concentrations of acetone and embedded in Epon resin. Ultrathin sections obtained in an ultramicrotome were stained with uranyl acetate and lead citrate and examined in a Zeiss 900 transmission electron microscope.

Flow Cytometry
The L. amazonensis axenic amastigotes (5 × 10 6 parasites/mL) were treated with 1 (50, and 100 µg/mL for 3 h at 32 °C), or untreated were harvested and washed with PBS. To gain analyses of mitochondrial membrane potential, (ΔΨm) parasites were stained with Rhodamine 123 (Rh 123) (5 mg/mL for 30 min at 37 °C) reagent following the protocol of manufacturer. The mean of fluorescence intensity of the cells was analyzed by flow cytometry FACSCalibur and CellQuest software). A total of 10,000 events were acquired in the region previously established as that corresponding to the parasites.

Measurement of Lipid Peroxidation Product
Samples of axenic amastigotes in exponential phase, were treated with 100 µg/mL of compound 1 for 6 h, at 32 °C. The thenoyltrifluoroacetone (TTFA) was used as positive control. After treatment, cells were washed with phosphate buffer, homogenized and added to a solution of 0.37% thiobarbituric acid in 15% trichloroacetic acid and 0.25 N HCl. The mixture was heated at 90-95 °C for 45 min. After cooling, butanol (1:1) was added to the solution. The mixture was shaken and centrifuged at 2000× g during 5 min. The optical density of the organic layer was determined at 535 nm in BIO-TEK Power Wave XS spectrophotometer. Lipid peroxidation was determined by the generation of thiobarbituric acid-reactive substances (TBARS) in terms of malondialdehyde (MDA), expressed in MDA nmol × protein mg −1 [41].

Statistical Analysis
All experiments were performed in duplicate. The means and standard deviations were determined from at least three experiments. Statistical analysis was performed with the program GraphPad Prism 4 (GraphPad Software, San Diego, California, USA). The student's t test was applied, and a p value less than 0.05 were regarded as significant.

Conclusions
New antileishmanials from natural products are urgently needed due to the emergence of drug resistance in patients. In this context, the use of the compound 1 isolated from C. cervicornis against L. amazonensis parasites is of great interest. The in vitro treatment of the parasites with compound 1 showed notable ultra structural changes, displayed depolarization in the mitochondrial membrane potential, and an increase of lipid peroxidation. Although the mechanism of action of the compound 1 is still unclear, these findings appear to be a future alternative to development of new antileishmanial drugs.