Activity of the Lupane Isolated from Combretum leprosum against Leishmania amazonensis Promastigotes

O presente trabalho descreve a atividade do extrato etanólico (EE) dos frutos de Combretum leprosum, do triterpeno 3b, 6b, 16b-triidroxilup-20(29)-eno (1) e seus derivados sintéticos (1a-1d), sobre promastigotas de Leishmania amazonensis. O EE apresentou atividade leishmanicida e o valor de IC50 foi de 24,8 mg mL. Já o triterpeno 3b, 6b, 16b-trihidroxilup-20(29)-eno (1), na concentração de 5,0 mg mL, apresentou uma potente ação inibitória sobre a proliferação das promastigotas (IC50 = 3,3 mg mL). Entre os derivados sintéticos, apenas 1b e 1d apresentaram atividade contra as promastigotas (IC50 = 3,48 mg mL e 5,8 mg mL, respectivamente). Por outro lado, o derivado sintético 1a não apresentou atividade sobre as promastigotas de L. amazonensis. O EE, (1) e os derivados sintéticos 1a-1d não apresentaram efeito citotóxico sobre macrófagos peritoneais de camundongos. Estes resultados fornecem evidencias de que o extrato etanólico e o lupano isolado de C. leprosum possui atividade contra promastigotas de L. amazonensis, podendo ser utilizados como ferramentas no estudo de novas drogas leishmanicidas.


Introduction
Leishmania, a protozoan parasite belonging to the family Trypanosomatidae, causes a broad spectrum of diseases, collectively known as leishmaniasis.Such conditions occur predominantly in tropical and subtropical regions.Approximately 350 million people live in areas of active Leishmania transmission, with 12 million people being directly affected by leishmaniasis in Africa, Asia, Europe and Americas. 1 Leishmania parasites have a complex life cycle that involves Phlebotomine sandfly vectors and mammalian hosts, with the amastigotes being within the phagolysosome of macrophages and the promastigotes in the vector's midgut. 2 The clinical manifestations of leishmaniasis are often divided in cutaneous, diffuse cutaneous, mucocutaneous and visceral leishmaniasis. 3,4Cutaneous leishmaniasis can Vol.22, No. 5, 2011   be spontaneously healed after a few months, or, depending on the Leishmania species, develop into diffuse cutaneous, relapsing cutaneous or mucocutaneous leishmaniasis.Visceral leishmaniasis, if untreated, leads to death in most patients. 5,6This disease causes considerable morbidity and severe face-disfigurement lesions on the affected people.
Nowadays, chemotherapy for leishmaniasis is still based on pentavalent antimonials (Glucantime and Pentostam), diamines (Pentamidine) and antifungal polyene (Amphotericin B).These are only a few of the drugs available since 1940.Unfortunately, they are generally toxic, expensive, share a tendency to generate resistance and require long-term treatments, which would make the conclusion complicated. 7Therefore, there is a great and urgent need to develop new, more effective and safer drugs for leishmaniasis control.

Instrumentation and chromatography
Silica gel (Merck 70-230 mesh) was used for all column chromatographies and solvents were redistilled prior to use. 1 H and 13 C NMR spectra 1D and 2D dimensions, were recorded at 300 and 75 MHz, respectively, using CDCl 3 or pyridine-d 5 , using TMS as internal standard; EIMS: Finnigan 3200 GC-MS instrument, electron impact mode, 70 eV.UV and IR spectra were obtained with a Perkin-Elmer PC FT-IR apparatus.

Ethanolic extract (EE) obtention, compound 1 isolation and purification
Dried fruits (2.7 kg) were powdered and then extracted with ethanol (5 L), being stirred and macerated at room temperature for approximately 24 h.This procedure was repeated three times.The solvent was fully evaporated under reduced pressure and the EE (58.3 g) was concentrated and stored at -20 ºC prior to use.Part of the EE (32.0 g) was submitted to coarse chromatography over silica gel (600 g) using hexane, CHCl 3 , EtOAc and MeOH as eluents.The fraction eluted with CHCl 3 was chromatographed on a silica gel column and was eluted with hexane-EtOAc, in increasing polarity.The fractions 27-30, eluted with hexane:EtOAc (30:70), were combined on the basis of thin layer chromatography (TLC) analysis and the presence of a white precipitate was observed, which after recrystallization from ethanol, was identified as 1 (2.37 g). 29

Preparation of the synthetic derivatives 1a-1d
The derivatives 1a-1d were prepared in accordance with Facundo et al. 29

Derivative 1a
0.54 mmol of 1 was dissolved in acetic anhydride (5.0 cm 3 ) and pyridine (1.0 cm 3 ).After 24 h, the material was poured over ice and the resulting mixture extracted with ethyl ether.The ethereal solution was washed with 3% aqueous HCl to eliminate the presence of pyridine.After solvent distillation and silica gel column purification, it yielded 0.34 mmol of the acetylated derivative 1a.

Derivative 1b
0.65 mmol of 1 was dissolved in dichloromethane (170 cm 3 ) and treated with pyridinium chlorochromate (1.53 mmol) for 2 h, under agitation, at room temperature.The mixture was filtered and extracted with ethyl ether solvent evaporation and silica chromatographic column purification, yielding 0.45 mmol of the oxidized product 1b.

Derivative 1c
0.22 mmol of 1a dissolved in dichloromethane (100 cm 3 ) was treated with pyridinium chlorochromate (0.65 mmol) for 2 h, under agitation.The mixture was filtered and extracted with diethyl ether.After solvent distillation and silica gel column purification, the crude product yielded 1c (0.17mmol).

Derivative 1d
0.10 mmol of 1c was dissolved in a saturated solution of methanol and hydroxide potassium.The mixture was kept under reflux for 3 h at 70 ºC.After organic solvent evaporation, 70 cm 3 of distilled water was added and the organic part was extracted with ethyl ether.After solvent evaporation and silica chromatographic column purification 0.08 mmol of 1d was obtained.
The structure of compound 1 and its synthetic derivatives (1a-d) are shown in Figure 1.The spectroscopic data are presented in the Supplementary Information.

Antileishmanial activity
The antiparasitic activity of ethanolic extract (EE), isolated triterpene 1 and synthetic derivatives (1a, 1b, 1d) from C. leprosum were evaluated against Leishmania amazonensis promastigote forms.The EE, 1 and the synthetic derivatives were dissolved in ethanol (less 1%).Promastigotes (5×10 5 per well) in 24-well were incubated in RPMI-1640 culture medium in the absence or in the presence of 1, 2 and 5 mg per mL of triterpenes and 12.5, 25, 50 and 100 mg per mL of the EE during 5 days at 23 o C. The number of living promastigotes was scored in the presence of erythrosin B. The results were expressed as the concentrations inhibiting parasite growth by 50% (IC 50 ) after a 5 days incubation period.Pentamidine was used as antileishmanial reference compound. 30

Toxicity on macrophages
Cell viability was assessed using a modified MTT assay. 31Briefly, elicited peritoneal mice macrophages (1×10 5 cells per well) were seeded in a 96-well plate and treated with 25 mg mL -1 of EE and 5 mg mL -1 of triterpenes.After 24 h, 10 μL of a MTT solution (5 mg mL -1 in phosphate buffered saline) was added to each well and furtherly incubated for 2 h at 37 °C.Subsequently, 100 μL of dimethylsulfoxide (DMSO) was added to each well to solubilize any deposited formazan and the optical density (OD) of each well was measured at 540 nm.

Statistical analysis
The inhibitory concentrations (ICs) were calculated by Probit Analysis using the program Minitab 14 (Minitab Inc).The statistical significance of group differences was evaluated using ANOVA and comparisons by Student-Newman-Keuls by the program SigmaStat (SPSS Inc, 1992-1997).

Results and Discussion
The pharmacological properties of Combretum leprosum have not been characterized up to this moment in details.Some studies had shown a promising potential for antinociceptive, anti-inflammatory, and antiulcerogenic activities. 32The arjunolic acid, isolated from roots and flowers of C. leprosum, displayed anti-inflammatory, antinociceptive and anticholinesterasic activities. 33riterpene, 3b,6b,16b-trihydroxylup-20(29)-ene (1), isolated from flowers, showed antinociceptive activity. 34n the present work we have shown an anti-leishmanicidal effect that had not been explored for this species yet.
The ethanolic extract (EE) of fruits of C. leprosum was tested in vitro against the promastigote forms of L. amazonensis.Quantifying Leishmania promastigote populational growth is involved in a number of investigations such as drug-efficacy testing and vaccine candidates. 35In such studies, the promastigote stage is most oftenly used once the promastigote is the infective form of the parasite and the proliferation capacity of a strain plays a key role in its infectivity potential. 36he EE showed an inhibitory activity, interfering in the promastigotes growth.A significant leishmanicidal effect was evident after 5 days of culture (Table 1).Promastigotes treated with 100 mg mL -1 of the EE or pentamidine died after 24 h of treatment.In all concentrations of EE (12.5-100 mg mL -1 ) studied, there was a significant difference in the growth of treated parasites compared to controls, not treated with the extract and treated with ethanol (F=318.6;P < 0.001).Promastigotes incubated with 1% (v/v) ethanol (the concentration necessary to dissolve the highest extract concentration used in the test) showed growth rates equivalent to the control cultures, indicating that the EE solvent was not toxic to the parasite.
The L. amazonensis promastigotes growth curve in the presence or absence of EE is shown in Figure 2. On the 5 th day of culture the promastigotes treated with the EE (25 mg mL -1 and 50 mg mL -1 ) showed a decrease in the parasite growth around 80% and 93%, respectively, compared with control or control treated with ethanol (Figure 2).The estimated IC 50 of EE for L. amazonensis promastigotes was 24.8 mg mL -1 .
Phytochemical studies carried out with some species of the genus Combretum indicated the presence of many classes of constituents, including triterpenes, flavonoids, lignans, non-protein amino acids, among others. 29,37,38McGaw et al. 16 using extracts of 20 species of Combretum, have shown that some of these, such as C. apiculatum, C. imberbe and  21,40,41 and C. micranthum have antiviral activity against herpes simplex virus types 1 and 2. 42 In order to investigate the role of 3b,6b,16btrihydroxylup-20(29)-ene (1), a lupane triterpene isolated from fruits of C. leprosum, we examined the effect of this compound in the L. amazonensis promastigotes growth.Various concentrations of this compound were used and the number of viable cells was scored every 24 h for up to 5 days.As shown in Figure 3A, after three days of culture, 5 mg mL -1 of compound 1 inhibited cell growth.The number of promastigotes reduced at least 80% at the fifth day of culture if compared to both controls; the one untreated and the other treated with ethanol.The estimated IC 50 of 1 for L. amazonensis promastigotes was 3.3 mg mL -1 (F = 1889.8;P < 0.001).This compound has a potent effect of inhibiting the promastigote growth.The others concentrations (2 and 1 mg mL -1 ) inhibited the parasite growth in 27% and 9% respectively, if compared to untreated control or ethanoltreated control (Figure 3A).
As another approach, in order to assess the biological activity and verify whether the role of structural modifications could increase or decrease biological activity, synthetic derivatives from this triterpene 1 (Figure 1) were prepared as described in Experimental.Derivative 1a, which had the hydroxyl groups at 3b and 16b positions Figure 3.In vitro effect of triterpene 1 isolated from C. leprosum fruits and its synthetic derivatives 1a-1d on parasite growth.Representative inhibition growth curves of L. amazonensis promastigotes incubated with a range of concentrations of natural triterpene 1 (A), derivative 1a (B), derivative 1b (C), and derivative 1d (D) during 5 days at 24 ºC.The promastigote growth inhibition was scored by counting of the viable parasites daily.The data shown is representative of at least five independent experiments.replaced by acetyl, became inactive (Figure 3B).Derivative 1b, that was trioxydated at positions 3b, 6b and 16b, had a similar antileishmanial activity if compared to the natural compound (Figure 3C).This derivative 1b at 5 mg mL -1 inhibited 80% of cell growth after 5 days of culture (F = 3951.8;P < 0.001).Incubation with 2 mg mL -1 of 1b inhibited 21% of parasite growth and 1mg mL -1 had no effect (Figure 3C).The estimated IC 50 of derivative 1b for L. amazonensis promastigotes was 3.48 mg mL -1 similar of natural compound.
The derivative 1d presents a carbonyl group at position C-6, differently from the natural triterpene 1, which has a hydroxyl group at this position.This derivative 1d also inhibited L. amazonensis growth in vitro at the concentration of 5 mg mL -1 , but the rate of inhibition was 41% (F = 402.98;P < 0.001).This compound interfered only slightly with parasite growth in culture.At the concentration of 2 mg mL -1 it inhibited only of 6.5% of promastigote growth (Figure 3D).The estimated IC 50 of derivative 1d was 5.9 mg mL -1 .
The derivative 1c that was monooxydated at positions 6b and had the hydroxyl groups at 3b and 16b positions replaced by acetyl was insoluble in ethanol, and therefore was not tested.The promastigotes incubated with ethanol (the vehicle used to dissolve the compounds 1 and 1a-1d) showed growth rates equivalent to ones of the control cultures, indicating that the solvent/vehicle was not toxic to the parasite.The promastigote culture treated with ethanol showed no significant difference compared to control.On the other hand there was a significant difference between the rate growth of natural triterpene-treated parasites and derivative 1a and 1d treated parasites at 5 mg mL -1 (P < 0.05).At this concentration the triterpene 1 and the synthetic derivative 1b have the same leishmanicidal activity, inhibiting promastigote growth in a similar pattern (Table 2).
Structural modifications in natural compound 1 allowed us to conclude that the acetyl groups at positions 3b and 16b led to inactivation of the lupane.But, the replacement of hydroxyl groups by carbonyl groups did not significantly affect its activity, as shown in the results from derivatives 1b and 1d.Compound 1 has IC 50 = 3.3 mg mL -1 , derivative 1b has IC 50 = 3.48 mg mL -1 and 1d, IC 50 = 5.8 mg mL -1 .
Screening compounds with known toxic effects against Leishmania and no effect against host cell is a useful approach in enhancing our knowledge of the biological events that regulate the processes of growth arrest and death in this parasite.By the way EE, the natural compound 1 and synthetic derivatives 1a-1d were evaluated for their cytotoxicity against mouse peritoneal macrophages and none of them were cytotoxic to the mammalian cells by MTT assay (Table 3).
In conclusion, the C. leprosum Mart.contains a bioactive triterpene, which has a significant activity against L. amazonensis promastigotes.The results presented in this paper furtherly support a new activity of triterpene 1 isolated from the fruits of C. leprosum and two of its synthetic derivatives 1b and 1d.The high pharmacological activity of 3b,6b,16b-trihydroxylup-20(29)-ene (1) from the C. leprosum fruits and the activity of the ethanolic extract on L. amazonensis promastigotes may be tools in further studies for the development of novel antileishmanial drugs.

Table 3 . 5
Effect of EE, 1 and 1a-1d on the cell mg mL -1 0.206 ± 0.018 Values are the mean ± SE of the three independent experiments.

Table 1 .
Growth inhibition of L. amazonensis cultures by the ethanolic extract (EE) of C. leprosum Mart. in vitro after five days .molle, present antiinflammatory activity; others, such as C. hereroense and C. paniculatum have antihelmint and antischistosoma activity, respectively.C. molle also has activity against Trypanosoma brucei rhodesiense and Plasmodium falciparum. 39Other Combretum species are capable of causing damages to the DNA of bacteria, such as C. apiculatum, C. mossambicense and C. hereroense.C. fragrans, C. padoides and C. erythrophyllum have antimicrobial activity against Gram-positive and Gramnegative bacteria One-way ANOVA, Student-Newman-Keuls (comparisons), n = 8 (mean ± SE).Different letters (a, b, c, d, e) indicate significant (P < 0.05) differences in a column.EE 100 mg mL -1 and Pentamidine killed all L. amazonesis promastigotes in culture after 5 days.C