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Antiparasitic activity and effect of casearins isolated from Casearia sylvestris on Leishmania and Trypanosoma cruzi plasma membrane.

ABSTRACT

Leishmaniasis and Chagas disease are infectious diseases caused by parasite Leishmania sp. and Trypanosoma cruzi, respectively, and are included among the most neglected diseases in several underdeveloped and developing countries, with an urgent demand for new drugs. Considering the antiparasitic potential of MeOH extract from leaves of Casearia sylvestris Sw. (Salicaceae), a bioguided fractionation was conducted and afforded four active clerodane diterpenes (casearins A, B, G, and J). The obtained results indicated a superior efficacy of tested casearins against trypomastigotes of T. cruzi, with [IC.sub.50] values ranging from 0.53 to 2.77 [micro]g/ml. Leishmania infantum promastigotes were also susceptible to casearins, with [IC.sub.50] values in a range between 4.45 and 9.48 [micro]g/ml. These substances were also evaluated for mammalian cytotoxicity against NCTC cells resulting in 50% cytotoxic concentrations ([CC.sub.50]) ranging from 1.46 to 13.76 [micro]g/ml. Additionally, the action of casearins on parasite membranes was investigated using the fluorescent probe SYTOX Green. The obtained results demonstrated a strong interaction of casearins A and B to the plasma membrane of T. cruzi parasites, corroborating their higher efficacy against these parasites. In contrast, the tested casearins induced no alteration in the permeability of plasma membrane of Leishmania parasites, suggesting that biochemical differences between Leishmania and T. cruzi plasma membrane might have contributed to the target effect of casearins on trypomastigotes. Thus, considering the importance of studying novel and selective drug candidates against protozoans, casearins A, B, G, and J could be used as tools to future drug design studies.

Keywords:

Casearia sylvestris

Salicaceae

Clerodane diterpenes

Leishmania

Trypanosoma cruzi

Casearins

Introduction

Casearia sylvestris a plant popularly known as "guafatonga", is geographically distributed throughout Latin America (Lorenzi and Matos 2002) and has been used in traditional medicine as anti-inflammatory, anti-ulcer, anti-ophidian and anti-tumor (Ferreira et al. 2011). The chemical composition of C. sylvestris has been characterized by clerodane type diterpenes, known as casearins A-X which showed pronounced antitumor activity (Itokawa et al. 1990; Morita et al. 1991; Carvalho et al. 1998; Wang et al. 2009a; Santos et al. 2010). Clerodane diterpenes with different stereochemistry from casearins, named casearvestrins A-C and other clerodane diterpenes have also been described from leaves of C. sylvestris (Oberlies et al. 2002; Santos et al. 2007; Wang et al. 2009b).

Protozoan diseases as Leishmaniasis and Chagas disease are main health and socioeconomic problems in many developing countries (WHO 2010). The chemotherapy for these diseases is unsatisfactory in terms of lack of effectiveness and also the undesirable side effects associated with long term treatment with discovered drugs (Schmidt et al. 2012). Medicinal plants have been used for the treatment of cutaneous leishmaniasis by rural people (Fournet et al. 1992; Franca et al. 1996) and have attracted more interest from the scientific community. Several compounds isolated from Brazilian plants have been described with potent anti- trypanossomal and antileishmanial activities (Schmidt et al. 2012). However, only eleven derivatives displayed comparable activity to standard drugs used in therapy to treat these tropical diseases, as could be seen in Table 1.

Mesquita et al. (2005) evaluated extracts of thirteen medicinal plants from the Brazilian Cerrado biome for antileishmanial (Leishmania donovani) and antitrypanosomal activities and discovered that the leaves extract of C. sylvestris exhibited activity at 100 [micro]g/mL However, no active compounds have been isolated so far.

Thus, the present work describes for the first time the antileishmanial and antitrypanosomal activities of casearins A, B, G, and J isolated from the MeOH extract from the leaves of C. sylvestris using bioguided fractionation procedures. Additionally, the effect on the permeability of plasma membrane of L. infantum and T. cruzi was evaluated.

Material and methods

General experimental procedures

[sup.1]H NMR and [sup.13]C spectra were recorded, respectively, at 300 and 75 MHz in a Bruker INOVA spectrometer. [CD.sub.3]OD (Aldrich) was used as the solvent and as the internal standard. Silica gel (Merck, 230-400 mesh), and Sephadex LH-20 were used for column chromatographic separation, while silica gel 60 [PF.sub.254] (Merck) was used for analytical and preparative thin layer chromatography.

Plant material

Leaves of C. sylvestris were collected from a single tree at the Atlantic Forest area in Sao Paulo city, SP, Brazil (coordinates 23 53'08.86"S, 46 40'10.45"O), in October, 2012. The identification was made by Dr. Oriana Favero (Universidade Presbiteriana Mackenzie-SP).

Extraction

Leaves of C. sylvestris (290 g), were dried and grounded, and then extracted using MeOH for 3 days, to afford 11.1 g of crude extract. The active MeOH extract was suspended in MeOH:[H.sub.2]O 2:1. After partition using hexanes, C[H.sub.2][Cl.sub.2] and EtOAc, were obtained the three phases which were evaluated to anti-trypanosomal and anti-Lesihmania activities.

Separation, purification and identification of chemical constituents

The active hexane phase (6.4g) was subjected to Si[O.sub.2] column chromatography gel eluted with increasing amounts of EtOAc in hexane (9:1 to 1:9) to give 23 fractions (A1-A23). The active fractions A11, A12 and A13 were submitted to new chromatographic steps guided by the antiparasitic activity. Thus, fraction A11 (380mg) was fractionated over Sephadex LH-20, eluted with MeOH and followed by Si[O.sub.2] prep TLC (hexanes 8: EtOAC 2) to afford caserins G (77 mg) andj (51 mg). Fraction A12 (300 mg) was also submitted to CC Sephadex LH-20 to yield casearin B (12 mg). Finally, fractionation of A13 (526 mg) over Sephadex LH-20, followed by prep TLC (hexanes 7: EtOAC 3) afforded casearin A (43 mg) (Fig. 1).

Parasite maintenance

L. (L.) infantum (MHOM/BR/1972/LD) was maintained in Golden hamsters, up to approximately 60-70 days post-infection. Promastigotes were maintained in M-l 99 medium supplemented with 10% calf serum and 0.25% hemin at 24[degrees]C. Trypomastigotes of T. cruzi (Y strain) were maintained in LLC-MK2 (ATCC CCL 7) cells using Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 2% calf serum at 37[degrees]C in a 5% C[O.sub.2] incubator (Tada et al. 1986).

Mammalian cells

The mammalian cells (NCTC--clone 929) and LLC-MK2 were maintained in M-199 medium without phenol red and supplemented with 10% fetal bovine serum at 37[degrees]C in a 5% C[O.sub.2] humidified incubator.

Evaluation of antiparasitic activity

In order to determine the 50% inhibitory concentration (IC50), the isolated casearins were dissolved in 100% MeOH, serially diluted in culture medium and incubated with parasites at concentrations in the range between 0.1 and 150 [micro]g/ml. The maximal concentration of MeOH in plate wells was 0.5% and internal controls were used to detect possible solvent toxicity to parasites.

Leishmania. The isolated casearins were incubated with promastigotes of L. (L.) infantum seeded at 1 x [10.s.up.6]/well in 96 well plates. After 48 h, the viability of promastigotes was measured by the mitochondrial activity using the MTT assay at 570 nm (Tada et al. 1986). Pentamidine was used as a positive control.

Trypanosoma cruzi. Trypomastigotes of T. cruzi (Y strain) obtained from LLC-MK2 cells were counted in a Neubauer hemocytometer and seeded at 1 x [10.sup.6]/well in 96-well microplates. Ater 24 h, the viability of trypomastigotes was measured by the mitochondrial activity using the MTT assay at 570 nm (Lane et al. 1996). Benznidazole was used as the standard drug.

Determination of the cytotoxicity against mammalian cells

The in vitro selectivity index ([CC.sub.50]/[IC.sub.50]) was determined using NCTC cells, which were seeded at 6 x [10.sup.4]/well in 96 well plates and M-l 99 medium as described above. The mammalian cells were incubated with casearins and the standard drugs pentamidine and benznidazole for 48 h at 37[degrees]C in a 5% C[O.sub.2] humidified incubator. The viability of cells was determined by the MTT assay as described above and the 50% cytotoxic concentration ([CC.sub.50]) was calculated.

Spectrofluorimetric detection of disruption of the parasites plasma membrane

Late growth-phase (non-stationary) promastigotes were washed in PBS, seeded at 2 x [10.sup.6]/well and incubated with 1 [micro]M SYTOX[R] Green for 15 min at 24[degrees]C (Mangoni et al. 2005). Casearins were added, and the fluorescence was measured every 20 min for 60 min total. The maximum permeabilization was obtained with 0.1% Triton X-100. Fluorescence intensity was determined using a fluorimetric microplate reader (FilterMax F5 Multi-Mode Microplate Reader-Molecular Devices) with excitation and emission wavelengths of 485 and 520 nm, respectively. The following internal controls were used in the evaluation: (i) the background fluorescence of casearins at the respective wavelengths, (ii) the possible interference of DMSO, (iii) untreated promastigotes, and (iv) medium without any cells. Samples were tested in triplicate.

Results

Isolation and identification of casearins

The chromatographic procedures carried out to active fractions A11, A12 and A13 allowed the isolation of four pure substances. The [sup.13]C NMR spectra of these fractions showed in general from 29 to 31 signals, similar to those reported for casearins in the literature. Characteristic signals in [sup.13]C NMR spectra were observed at [delta] 57 (CH3) indicating the presence of a methoxy group in the structure, and [delta] 72 (CH) for the carbinolic carbon (C2) in all isolated compounds. The signals at [delta] 75 (CH) were assigned to carbons attached to hydroxyl groups (C6), except to casearin G, which displayed the methylenic carbon (C6) at [delta] 34. The double bond at C-3 and C-4 also characteristic of casearins was observed at [delta] 123 and 142 respectively. The chemical shifts at [delta] 53 (C-5) and [delta] 35 (C-10) were similar to those described for casearins and analogous compounds. The signals of methynic carbons at [delta] 95 (C-18) and 97 (C-19) indicate the existence of the diacetalic ring system C, while those at [delta] 170-174 suggest the presence of acetate and butanoate groups as substituents.

Determination of the 50% inhibitory concentration and mammalian cytotoxicity

Casearins were incubated for 24 h with T. cruzi trypomastigotes and 48 h with promastigotes of L (L.) infantum. The viability was determined by the colorimetric assay of MTT. All tested substances killed 100% of parasites at the highest tested concentration (150 [micro]g/ml); casearins A, B, G and J showed [IC.sub.50] values in a range between 0.53 and 2.7 [micro]g/ml against T. cruzi trypomastigotes (Table 2). The most promising activity was observed for casearin J, which showed an [IC.sub.50] value of 0.53 [micro]g/mL. Leishmania promastigotes were also susceptible to casearins resulting in [IC.sub.50] values ranging from 4 to 9 [micro]g/ml (Table 2). Similarly to T. cruzi assay, the most active substance against Leishmania was casearin J, which showed an [IC.sub.50] value of 4.45 [micro]g/ml (Table 2). Casearins were also tested for cytotoxicity after incubation with NCTC cells for 48 h. The viability was also determined using the MTT assay. Casearins showed [CC.sub.50] values in a range between 1 and 13 [micro]g/ml (Table 2). It was also possible to note that casearin J was the most cytotoxic substance.

Plasma membrane permeabilization

The activity of casearins on parasites membrane of trypomastigotes and promastigotes was evaluated using the fluorescent probe SYTOX green (Fig. 2). The isolated casearins resulted in an intense and time-dependent increase in fluorescence levels when incubated with T. cruzi parasites. When compared to fully permeabilized (100%) parasites, casearins showed the following percentage of permeability alteration on T. cruzi membranes after 60 min incubation: (i) casearin J = 29%; (ii) casearin G = 58%; (iii) casearin B = 80% and (iv) casearin A = 87%. According to ANOVA test, the difference among the tested substances was significant (p < 0.001). Methanol at 0.5% was used as internal control and showed no alteration in permeability of membranes. Casearins showed minimum alteration in the permeability of Leishmania membrane when compare to fully (100%) permeabilized parasites after 60 min, as follows: (i) casearin J = 6%; (ii) casearin G = 2.8%: (iii) casearin B = 4.6% and (iv) casearin A = 4%. According to ANOVA test, the difference among the tested substances was significant (p < 0.001).

Discussion

Clerodane diterpenes have been isolated from different plant species such as Baccharis tricuneata (Wagner et al. 1978), Zuelania guidonia (Khan et al. 1990), Laetia procera (Jullian et al. 2005) and Laetia corymbulosa (Beutler et al. 2000). These derivatives are characteristic of the genus Casearia and have been previously described from several species such as Casearia rupestris (Vieira-Junior et al. 2011), Casearia obliqua (Vieira-Junior et al. 2009) and Casearia nigrencens (Williams et al. 2007). However, characteristic diterpenes from C. sylvestris are known as caseaerins/casearvestrins and display promising biological activities, such as chemopreventive effect and anti-tumor action (Prieto et al. 2013; Ferreira et al. 2010). The structural identification of casearins A, B, G and J, isolated in this work was performed by comparing the data obtained from the spectral [sup.1]H and [sup.13]C NMR with the literature data (Itokawa et al. 1990; Morita et al. 1991; Carvalho et al. 1998).

Folk uses and scientific investigations have highlighted the importance of C. sylvestris extracts and their relevant bioactive potential, as anti-ulcer, anti-inflammatory, anti-ophidian and antitumor potentialities (Ferreira et al. 2011). Previous studies reported the antileishmanial and antitrypanosomal activity of crude extracts from Brazilian Cerrado plants, including C. sylvestris (Mesquita et al. 2005), but no information about the activity of isolated compounds have been described. Thus, this work demonstrates for the first time the in vitro activity of casearins A, B, G and J, isolated from C. sylvestris against T. cruzi and Leishmania parasites. Our data demonstrated that tested casearins were most effective against T. cruzi parasites in comparison to standard drug benznidazole. Although casearin J showed the most promising activity against T. cruzi, casearins B and G were the most selective substances, with a selectivity index ([CC.sub.50] mammalian cells/[IC.sub.50] parasite) determined as approximately 5. Considering the structural differences among the isolated casearins, we suggest that the substituent butanoate at C-18 present in casearin J might have contributed to the higher efficacy among the four tested diterpenoids. Despite no antitrypanosomal activity of casearins has previously been described in the literature, a related clerodane diterpene isolated from C. sylvestris showed a similar activity on T. cruzi, with minimal inhibitory concentration (MIC) at 0.59 [micro]g/mL (Espindola et al. 2004).

Leishmania parasites were also susceptible to tested casearins, but to a lesser extent when compared to T. cruzi. According to the analysis of the [IC.sub.50] values, tested casearin resulted in a lower efficacy than the standard drug pentamidine. Similarly to the activity observed against T. cruzi, casearin J also demonstrated the highest efficacy among the tested substances against Leishmania, corroborating our above cited suggestions about the structure-activity relations. As a consequence of the elevated toxicity to mammalian cells and weak antileishmanial activity, casearins were tested only against the extracellular form of Leishmania. To date, this is also the first report describing the antileishmanial activity of casearins.

Targeting protozoan plasma membranes has been considered a promising approach to the study of novel drug candidates (Oliveira et al. 2012). When one considers the differences between mammalian plasma membrane and those of protozoan parasites, it is possible to envisage biochemical targets for drugs. Sterols are the major structural components of cell membranes and stabilize their structure by interacting with the fatty acyl moieties of membrane phospholipids to affect membrane fluidity. The sterol contents of microorganisms such as fungi and parasites of the Trypanosomatidae family differ from those of mammalian cells because of the predominant presence of ergosterol, episterol, and other 24-methyl sterols, which are completely absent in the host cells (Medina et al. 2012). Thus, the effect of casearins on the permeability of T. cruzi and Leishmania plasma membranes was evaluated using the fluorescent probe SYTOX green. Our data clearly showed that casearins have a higher affinity to T. cruzi membrane than Leishmania, suggesting a time-dependent event that might have contributed to cell death. Membrane disruption results in depletion of ionic gradients across the plasma membrane, efflux of nutrients and other cytoplasmic components, breakdown of parasite bioenergetic system and osmotic lysis, resulting in cell death (Marr et al. 2012). Opposing to the observed antitrypanosomal effect, in which casearin J presented the highest antiparasitic activity, it is clear that this diterpene induced a minimum alteration of the plasma membrane of T. cruzi when compared to other tested casearins. Among these substances, our assays demonstrated that casearins A and B showed the highest affinity to plasma membrane of T. cruzi, causing a strong permeabilization, which may have contributed to parasite death. The presence of the acetate substituent at C18 in addition of oxygenated C6 on the structure of casearins A and B may also have led to a superior affinity to T. cruzi membrane. Although the tested casearins showed antileishmanial activity against promastigotes, no alteration in the permeability of the plasma membrane was observed, suggesting a different lethal action on Leishmania parasites, which might be exploited for future studies.

Conclusion

The obtained results indicated that the tested casearins cause alteration in the permeability of plasma membrane of T. cruzi. Therefore, considering the importance of studying novel and selective drug candidates against protozoans, casearins could be used as promising tools to future drug design studies.

http://dx.doi.org/10.1016/j.phymed.2014.01.004

ARTICLE INFO

Article history:

Received 3 September 2013

Received in revised form 25 October 2013

Accepted 14January 2014

Acknowledgments

The authors thank FAPESP (2011/51739-0 and 2013/16320-4) and CNPq (470853/2012-3) for financial support for the development of this work. We also thank CAPES and FAPESP fellowships to DDB and EGP, respectively, and CNPq for the scientific research award to JF1GL and ACT. The authors wish to thank Mr. Carlos R. Figueiredo for the collection of Casearia sylvestris.

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WHO, 2010. Working to Overcome the Global Impact of Neglected Tropical Diseases: First WHO Report on Neglected Tropical Diseases. WHO, Geneva, pp. 184.

Williams, R.B., Norris, A., Miller, J.S., Birkinshaw, C., Ratovoson, F., Andriantsiferana, R., Rasamison, V.E., Kingston, D.G.I., 2007. Cytotoxic clerodane diterpenoids and their hydrolysis products from Casearia nigrescens from the rainforest of Madagascar. J. Nat. Prod. 70, 206-209.

Diego Dinis Bou (a), Andre G. Tempone (b), Erika G. Pinto (b,c), Joao Henrique G. Lago (a), Patricia Sartorelli (a), *

(a) Instituto de Ciencias Ambientais, Qutmicas e Farmaceuticas, Universidade Federal de Sao Paulo. 09972-270 Diadema, SP, Brazil

(b) Departamento de Parasitologia, Instituto Adolfo Lutz, 01246-000 Sao Paulo, SP, Brazil

(c) Instituto de Medicina Tropical de Sao Paulo, Universidade de Sao Paulo, Av. Dr. Eneas de Carvalho Aguiar, 470, 05403-000 Sao Paulo, SP, Brazil

* Corresponding author. Tel: +55 11 3319 3300; fax: +55 11 4043 6428.

E-mail address: psartorelli@unifesp.br (P. Sartorelli).

Table 1
Brazilian plants and isolated compounds which displayed antiparasitic
activity (anti-trypanossomal and antileishmanial) and comparison of
IC5o values with those reported to positive controls.

Scientific name           Parasite form
(family)

Aspidosperma ramiflorum   L amazonensis
  (Apocynaceae)             (promastigotes)
Drimys brasiliensis       T. cruzi
  (Winteraceae)             (trypomastigotes)
Nectandra megapotamica    T. cruzi
  (Lauraceae)               (trypomastigotes)

Peschiera australis       L amazonensis
  (Apocynaceae)             (amastigotes)
Piper regnelli            L amazonensis
  (Piperaceae)              (amastigotes)
Piper solmsianum          T. cruzi
  (Piperaceae)              (trypomastigotes)

Tanacetum parthenium      L amazonensis
  (Asteraceae)              (promastigotes)
Virola surinamensis       T. cruzi
  (Myristicaceae)           (trypomastigotes)

Scientific name           [IC.sub.50] values
(family)
                          Active compounds

Aspidosperma ramiflorum   Ramiflorine B (4.9 [micro]g/ml)
  (Apocynaceae)
Drimys brasiliensis       Polygodial (2.03 [micro]g/ml)
  (Winteraceae)
Nectandra megapotamica    Machilin G (0.78 [micro]g/ml)
  (Lauraceae)             Galgravin (1.64 [micro]g/ml)
                          Calopiptin (4.49 [micro]g/ml)
                          Ganschisandrine (4.54 [micro]g/ml)
Peschiera australis       Coronaridine (4.7 [micro]g/ml)
  (Apocynaceae)
Piper regnelli            Eupomatenoid-5 (5.0 [micro]g/ml)
  (Piperaceae)
Piper solmsianum          3,4,3',4'-Dimethylenedioxy-
  (Piperaceae)            5,5'-dimethoxy-7,7'-
                          epoxylignan
                          (3.47 [micro]g/ml)
Tanacetum parthenium      Parthenolide (0.37 [micro]g/ml)
  (Asteraceae)
Virola surinamensis       Grandisin (1.5 [micro]g/ml)
  (Myristicaceae)

Scientific name           [IC.sub.50] values
(family)
                          Positive controls

Aspidosperma ramiflorum   Pentamidine (10.0 [micro]g/ml)
  (Apocynaceae)
Drimys brasiliensis       Benznidazole (38.3 [micro]g/ml)
  (Winteraceae)
Nectandra megapotamica    Gentian violet (31.0 [micro]g/ml)
  (Lauraceae)
Peschiera australis       Glucantime (6.6 [micro]g/ml)
  (Apocynaceae)
Piper regnelli            Anphotericin (0.23 [micro]g/ml)
  (Piperaceae)
Piper solmsianum          Gentian violet (31.0 [micro]g/ml)
  (Piperaceae)
Tanacetum parthenium      --
  (Asteraceae)
Virola surinamensis       Gentian violet
  (Myristicaceae)

Scientific name           Reference
(family)

Aspidosperma ramiflorum   Tanaka et al. (2007)
  (Apocynaceae)
Drimys brasiliensis       Correa et al. (2011)
  (Winteraceae)
Nectandra megapotamica    Silva Filho et al. (2004)
  (Lauraceae)
Peschiera australis       Delorenzi et al. (2001)
  (Apocynaceae)
Piper regnelli            Vendrametto et al. (2010)
  (Piperaceae)
Piper solmsianum          Martins et al. (2003)
  (Piperaceae)
Tanacetum parthenium      Tiuman et al. (2005)
  (Asteraceae)
Virola surinamensis       Lopes et al. (1998)
  (Myristicaceae)

Table 2
Antileishmanial, antitrypanosomal and cytotoxicity effects of
casearins isolated from C. sylvestris.

Casearins    [IC.sub.50] ([micro]g/ml)   [IC.sub.50] ([micro]g/ml)
             promastigotes               trypomastigotes
             L (L) infantum              T. cruzi
             95% CI                      95% CI

A            6.34 * (4.01-10.01)         1.37 * (1.07-1.75)
B            9.48 * (7.43-12.09)         2.77 * (2.63-2.93)
J            4.45 * (3.93-5.04)          0.53 * (0.35-0.81)
G            5.93 * (5.59-6.29)          1.57 * (1.23-2.00)

Casearins
             [CC.sub.50] ([micro]g/ml)
             Cell NCTC
             95% CI

A             2.62 (1.92-3.59)
B            13.76 (9.19-20.58)
J             1.46 (1.36-1.58)
G             7.54 (5.84-9.74)

95% CI = 95% confidence interval; Leishmania (promastigotes)--
pentamidine [IC.sub.50] 0.22 [micro]g/ml; T. cruzi (trypomastigotes)--
benznidazole [IC.sub.50] 114.68 [micro]g/ml. * p < 0.05 (compared to
standard drug).
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Title Annotation:Infections
Author:Bou, Diego Dinis; Tempone, Andre G.; Pinto, Erika G.; Lago, Joao Henrique G.; Sartorelli, Patricia
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Apr 15, 2014
Words:4974
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