Drimanes from Drimys brasiliensis with leishmanicidal and antimalarial activity

This paper evaluates CHCl 3 and CH 3 OH extracts of the stem bark, branches and leaves of Drimys brasiliensis and drimane sesquiterpenes isolated from the stem bark against strains of Leishmania amazonensis and Leishmania braziliensis promastigotes and Plasmodium falciparum trophozoites. All of the extracts and compounds were tested in cell lines in comparison with reference standards and cell viability was determined by the XTT method. The CHCl 3 and CH 3 OH extracts from the stem bark and branches yielded promising results against two strains of Leishmania , with 50% inhibitory concentrations (IC 50 ) values ranging from 39-100 µg/mL. The CHCl 3 extract of the stem bark returned IC 50 values of 39 and 40.6 µg/mL for L. amazonensis and L. braziliensis , respectively. The drimanes were relatively effective: 1-β-(p-coumaroyloxy)-polygodial produced IC 50 values of 5.55 and 2.52 µM for L. amazonensis and L. braziliensis , respectively, compared with 1-β-(p-methoxycinnamoyl)-polygodial, which produced respective IC 50 values of 15.85 and 17.80 µM. The CHCl 3 extract demonstrated activity (IC 50 of 3.0 µg/mL) against P. falciparum . The IC 50 values of 1-β-(p-cumaroyloxyl)-polygodial and 1-β-(p-methoxycinnamoyl)-polygodial were 1.01 and 4.87 µM, respectively, for the trophozoite strain. Therefore, the results suggest that D. brasiliensis is a promising plant from which to obtain new and effective antiparasitic agents.

Neglected tropical diseases, including leishmaniasis, are among the most common causes of illness among the poorest populations living in developing countries.Cutaneous leishmaniasis has a worldwide distribution and is a public health problem, with 1-1.5 million cases recorded annually worldwide (Hotez et al. 2008).The World Health Organization (WHO) considers leishmaniasis to be one of six major infectious diseases, with a high detection rate and the capacity to produce deformities.It is a disease with a variety of agents, hosts, vectors and transmission patterns, but limited knowledge about some aspects of the disease makes it difficult to control (WHO 2010).
Another serious public health problem is malaria, which is caused by protozoa of the genus Plasmodium and is transmitted to humans by female Anopheles mosquitoes, producing fever and other symptoms.In Brazil, the greatest number of cases is recorded in the Amazon Region, where the environmental and sociocultural conditions favour the expansion of plasmodial transmission (WHO 2009).
The intense search for new ways of treating these neglected diseases has motivated scientific research in this field.The importance of parasitic diseases as a public health issue, coupled not only with the limited number of drugs available for their treatment, but also their significant side effects, has led to the important strategy of searching for new antiparasitic agents among natural or synthetic compounds.
It has recently been demonstrated that a hexane extract of the stem bark of Drimys brasiliensis and polygodial, a drimane, exhibit activity against Leishmania spp and Trypanosoma cruzi (Corrêa et al. 2011).This information contributes to the further study of this plant species, with the aim of obtaining new drugs to treat the neglected diseases.This paper analyses chloroform and methanol extracts of the stem bark, branches and leaves of D. brasiliensis and selected pure drimane sesquiterpenes for their inhibitory activity against strains of Leishmania amazonensis and Leishmania braziliensis promastigotes and Plasmodium falciparum trophozoites.

MATERIALS AND METHODS
General experimental procedures -NmR spectra for 1 H (300 mHz) and 13 C (75.5 mHz) were obtained on a Varian Gemini AC 300 spectrometer with CDCl 3 as the solvent.The chemical shifts are given in δ (ppm) with TmS as the internal standard.Silica gel 60 (230-400 mesh) was used for the column chromatography and silica gel 60 F 234 (230-400 mesh) was used for the thin layer chromatography.
Plant material -The stem bark, branches and leaves of D. brasiliensis were collected in Rancho Queimado, in the Brazilian state of Santa Catarina (SC), in December 1998.The plant was identified as D. brasiliensis miers subsp.sylvatica (Saint Hilaire) Ehrendofer & Gottsb by Dr Ademir Reis (Department of Botany, Federal University of Santa Catarina) and a voucher specimen was compared with one deposited in the Barbosa Rodrigues Herbarium (Itajaí, SC), under the protocol VC Filho 010.
Preparation of extracts -The stem bark of D. brasiliensis (774 g) was dried at 40ºC for two days, powdered and successively extracted with chloroform and CH 3 OH at room temperature for seven days each.The extracts were concentrated under reduced pressure, yielding residues of 30.2 and 60.6 g, respectively.The branches (700 g) were processed in the same way as the bark to produce a yield of 17.6 and 14.8 g from the CHCl 3 and meOH extracts, respectively.From 250 g of leaves, the same method yielded 24.5 and 10.6 g of material from the CHCl 3 and meOH extracts, respectively.
Isolation and identification of compounds from the CHCl 3 bark extract -An aliquot of the stem bark extract (25 g) was chromatographed (θi 5.4 cm) on a column that was packed with silica gel (238 g) and the extract components were eluted with hexane gradually enriched with ethyl acetate and ethanol.Seventy fractions of 100 mL each were collected.Spathulenol (Figure, compound 1) was obtained from fractions 4-21 [elution solvent Hex-AcOEt (9:1)] with a yield of 603 mg (0.0645% of the dry plant material).Polygodial (Figure,compound 2) in the amount of 732.8 mg (0.078% of the dry plant material) was obtained from fractions 26-30 [elution solvent Hex-AcOEt (9:1)].
Isolation and identification of compounds from the CHCl 3 branch extract -An aliquot of the branch extract (13.3 g) was chromatographed on a column (θi 3,5 cm) packed with silica gel (100 g) and the adsorbent was eluted with hexane gradually enriched with ethyl acetate and ethanol.Seventy-five fractions of 100 mL each were collected.Using this procedure, only mixtures of fatty acids and alcohols were obtained.
Isolation and identification of compounds from the CHCl 3 leaf extract -Part of the leaf extract (17.1 g) was subjected to chromatography (θi 4.0 cm) on a column packed with silica gel (150 g) and the adsorbent was eluted with hexane gradually enriched with ethyl acetate and ethanol.Fifty-five fractions of 100 mL each were collected.Fraction 34 collected in hexane:ethyl acetate at a 1:1 ratio (1.134 g) was chromatographed (θi 2,0 cm) on a column packed with silica gel (27 g) and eluted with hexane gradually enriched with ethyl acetate and ethanol.Thirty-five subfractions of 35 mL each were collected.Fractions 12-16, which were eluted with hexane:ethyl acetate at an 8:2 ratio, yielded 157.4 mg of a mixture of α and β-cubebin (0.0438% of the dry plant material).
All of the compounds were identified based on their spectral data through comparison with the literature (malheiros et al. 2001) and directly with authentic samples.µL) and counted in a Neubauer chamber.The population was adapted to 3 x 10 6 parasites/mL with Schneider medium (pH 6.8) and foetal bovine serum (10%) distributed (100 µL/well) in 96-microwell plates.Solutions of the samples to be assessed at final concentrations of 100, 50 and 25 µg/mL were added (100 µL) to the wells of the plates with the parasites.Dimethyl sulfoxide (DmSO) (1%) and amphotericin B (0.5 µg/mL) were used as parasite growth controls.Each test was performed in triplicate and the plates were incubated for 72 h at 26ºC.

Biological activity assays -
Then, 50 µL of a solution of XTT (1 mg/mL) in phosphate buffer (pH 7.0, 37ºC) with phenazine methosulfate (Sigma-Aldrich, 0.06 mg/mL) was added to each well of the plates, which were incubated for another 4 h at 26ºC.The plates were read on a Stat Fax 2100 microplate reader at 450 nm.The 50% inhibitory concentrations (IC 50 ) values for the parasites were calculated using microsoft Excel 2000 (Capusiri et al. 2008).
Antiplasmodial activity -The P. falciparum parasites belonged to the F32-Tanzania strain (chloroquine-sensitive, kindly provided by Dr T Fandeur, Pasteur Institute, Kayenne) and were cultivated under anaerobic conditions at 37ºC in RPmI-1640 medium supplemented with 10% human serum containing erythrocytes at a haematocrit of 4% (blood group O, RH + ) (Trager & Jensen 1976).The parasites were synchronised at 1% parasitaemia and 2% haematocrit before their distribution into 96-well plates at a volume of 100 µL in duplicate.A total of 100 µL of each concentration of each compound or extract (10, 1 or 0.1 µg/mL in DmSO -final concentrations did not exceed 0.1%) was added to each well and chloroquine (100 nm) was used as a control.The parasites were then incubated at 37ºC for 48 h.Next, a smear of the parasites was fixed with methanol and stained with Giemsa.The antiplasmodial activity was determined by examining the slides under a microscope and counting the non-infected red cells (GRL) and infected cells (GRI) to obtain the inhibition percentage (formula 1).
% Inhibition = (GRL -GRI) x 100 (1) GRL The IC 50 for schizont ripening was determined by a graphic method using the program CRIKET GRAPH 1.3 and those treatments with IC 50 values < 10 µg/mL were considered to have inhibitory activity.
Statistical analysis of data -The samples were analysed in replicates with n = 4 using two methods to assess viability (mTT and neutral red).The data are reported as means ± standard errors and the significant differences among the treatments were determined using a one-way ANOVA followed by the Tukey-Kramer test (*p < 0.05, **p < 0.01 and ***p < 0.001).

RESULTS
In the search for new potential antiparasitic agents, the chloroform and methanol extracts of the stem bark, branches and leaves and the isolated compounds from the stem bark of D. brasiliensis miers were assessed in vitro for their capacity to inhibit development of the promastigotes of L. amazonensis and L. brasiliensis and the trophozoites of P. falciparum.The resulting minimal IC are shown in Tables I, II.
For the initial screening of substances inhibiting the Leishmania spp, those extracts with IC 50 values less than 50 µg/mL were considered active.Thus, the IC 50 values of 39.0 and 40.6 µg/mL for the stem bark chloroform extract tested against L. amazonensis and L. brasiliensis, respectively, indicated the leishmanicidal activity of the extract (Table I).The methanol and chloroform extracts of the branches presented IC 50 values between 78.0-99.0µg/mL, against the promastigote forms of the test Leishmania and the leaf extracts were inactive.
It is possible to compare these results with those reported in the literature for various plant extracts.For example, extracts of Bidens pilosa L. and Punica granatum L. inhibited the growth of intracellular amastigotes, with IC 50 values of 42.6 and 69.6 µg/mL, respectively.Extracts of Julocroton triqueter, a Dichorisandra sp. and Tephrosia cinerea were shown to be more effective in inducing the death of promastigotes, with IC 50 values of 29.5, 32.9 and 43.6 µg/mL, respectively (Luize et al. 2005, Rocha et al. 2005, Bezerra et al. 2006, García et al. 2010).

DISCUSSION
Based on the results presented above, D. brasiliensis can be considered a source of compounds with leishmanicidal activity.The chloroform extracts of the stem bark, branches and leaves were subjected to column chromatography.The fractionation of the chloroform extract of the stem bark led to the isolation of five compounds, which were identified as spathulenol ( The column chromatography of the chloroform extracts yielded the isolation of principally fatty alcohols and acids from the branches and fatty alcohols and acids and a mixture of the lignans α and β-cubebin from the leaves.The compounds were characterised by H 1 and C 13 NmR and comparison of the NmR spectra with previously reported data (malheiros et al. 2001, Lago & Roque 2009).
Considering that the stem bark extract was rich in drimane sesquiterpenes and exhibited excellent inhibitory activity when evaluated in vitro against the promastigotes of L. amazonensis and L. brasiliensis, the drimanes were evaluated to identify the compound or compounds responsible for this inhibition.All of the drimanes exhibited activity against the promastigotes, with IC 50 values < 42 µM.The compound 1-β-(p-cumaroyloxyl)-polygodial (Figure,compound 5) exhibited the best activity, with IC 50 values of 5.55 and 2.52 µm, respectively, against L. amazonensis and L. brasiliensis.Amphotericin B, which was used as a positive control, activity against L. brasiliensis and had an IC 50 of 1.1 µm.
Comparing the drimanes with each other, those with a cinnamoyl group at the C-1 position exhibited higher activity than did polygodial, which lacks this group.This increased activity can be related to the lipophilicity caused by the addition of the aromatic ring to these compounds.moreover, the substitution of a hydroxycinnamoyl group (Figure,compound 5) for the methoxycinnamoyl group (Figure,compounds 3,4) led to increased leishmanicidal activity against the promastigotes of both of the Leishmania spp analysed.These results highlight the importance of the p-hydroxycinnamoyl group for the leishmanial inhibition, which may be explained by the lipophilicity associated with the aromatic group and the possibility of hydrogen bond formation to maintain the biological activity.Another compound evaluated was the aromadendrane spathulenol (Figure, compound 1), which was isolated from the chloroform extracts.As can be seen from the data in Table I, spathulenol did not contribute to the activity of this extract.
The extracts obtained from the different parts (the stem bark, branches and leaves) of D. brasiliensis were also evaluated for their antiplasmodial activity.The initial screening revealed plasmodicidal activity only for the chloroform extract of the stem bark, which produced an IC 50 value of 3.0 µg/mL and 97% inhibition (Table II).
Our results, together with those reported in the literature, strongly suggest that D. brasiliensis and the drimanes could be promising for the treatment of diseases such as leishmaniasis and malaria, two of the so-called neglected diseases caused by protozoans, which demand the search for new chemotherapeutic agents.However, further studies (in vitro and in vivo) are imperative for understanding the mechanisms of action of these bioactive compounds and for evaluating their toxicity to humans before these agents can be exploited clinically.
This paper evaluates CHCl 3 and CH 3 OH extracts of the stem bark, branches and leaves of Drimys brasiliensis and drimane sesquiterpenes isolated from the stem bark against strains of Leishmania amazonensis and Leishmania braziliensis promastigotes and Plasmodium falciparum trophozoites.All of the extracts and compounds were tested in cell lines in comparison with reference standards and cell viability was determined by the XTT method.The CHCl 3 and CH 3 OH extracts from the stem bark and branches yielded promising results against two strains of Leishmania, with 50% inhibitory concentrations (IC 50 ) values ranging from 39-100 µg/mL.The CHCl 3 extract of the stem bark returned IC 50 values of 39 and 40.6 µg/mL for L. amazonensis and L. braziliensis, respectively.

TABLE I
Leishimanicidal activity of extracts and compounds isolated from stem bark of Drimys brasilensis in vitro on promastigote forms of Leishmania amazonensis and Leishmania brasiliensis data are expressed as mean standard deviation of three determinations.IC 50 : 50% inhibitory concentrations.

TABLE II Plasmodicidal
activity of extracts and compounds isolated from stem bark of Drimys brasilensis in vitro on trophozoite forms of Plasmodium falciparum 50 : 50% inhibitory concentrations.