Unlocking the in vitro anti-Trypanosoma cruzi activity of halophyte plants from the southern Portugal

Marta Oliveira， Policarpo Ademar Sales Junior， Maria Jo?o Rodrigues， Marina DellaGreca，Luísa Barreira， Silvane Maria Fonseca Murta， Alvaro José Romanha， Luísa Custódio?

1Center of Marine Sciences （CCMAR）， University of Algarve， Campus de Gambelas 8005-139 Faro， Portugal

2Centro de Pesquisas Rene Rachou， FIOCRUZ， Belo Horizonte， Minas Gerais， Brazil

3Dipartimento di Chimica Organica e Biochimica， Universita` Federico II， Complesso Universitario Monte Sant'Angelo， Via Cintia 4， I-80126，Napoli， Italy

Unlocking the in vitro anti-Trypanosoma cruzi activity of halophyte plants from the southern Portugal

Marta Oliveira1， Policarpo Ademar Sales Junior2， Maria Jo?o Rodrigues1， Marina DellaGreca3，Luísa Barreira1， Silvane Maria Fonseca Murta2， Alvaro José Romanha2， Luísa Custódio1?

1Center of Marine Sciences （CCMAR）， University of Algarve， Campus de Gambelas 8005-139 Faro， Portugal

2Centro de Pesquisas Rene Rachou， FIOCRUZ， Belo Horizonte， Minas Gerais， Brazil

3Dipartimento di Chimica Organica e Biochimica， Universita` Federico II， Complesso Universitario Monte Sant'Angelo， Via Cintia 4， I-80126，Napoli， Italy

ARTICLE INFO

Article history：

Received 15 May 2016

Received in revised form 16 June 2016

Accepted 15 July 2016

Available online 20 August 2016

Chagas disease

Halophytes

Trypanosoma cruzi

Phenanthrenes

Juncunol

Objective： To evaluate the in vitro anti-Trypanosoma cruzi （T. cruzi） activity of organic extracts prepared from halophyte species collected in the southern coast of Portugal （Algarve）， and chemically characterize the most active samples. Methods： Acetone， dichloromethane and methanol extracts were prepared from 31 halophyte species and tested in vitro against trypomastigotes and intracellular amastigotes of the Tulahuen strain of T. cruzi. The most active extract was fractionated by preparative HPLC-DAD， affording 11 fractions. The most selective fraction was fully characterized by1H-NMR. Results： From 94 samples tested， one was active，namely the root dichloromethane extract of Juncus acutus （IC50＜ 20 μg/mL）. This extract was fractionated by HPLC， affording 11 fractions， one of them containing only a pure compound（juncunol）， and tested for anti-parasitic activity. Fraction 8 （IC50= 4.1 μg/mL） was the most active，and was further characterized by1H-NMR. The major compounds were phenanthrenes， 9，10-dihydrophenanthrenes and benzocoumarins. Conclusion： Our results suggest that the compounds identified in fraction 8 are likely responsible for the observed anti parasitic activity. Further research is in progress aiming to isolate and identify the specific active molecules. To the best of our knowledge， this is the first report on the in vitro anti T. cruzi activity of halophyte species.

1. Introduction

Chagas disease （CD） is a neglected tropical disease （NTD） caused by the protozoan parasite Trypanosoma cruzi （T. cruzi）， transmitted to humans and animals from the faeces of triatomine bugs （kissingbugs）. It is estimated that 20% to 30% of humans infected with T. cruzi suffer with severe cardiopathy or megaesophagus -megacolon ［1］. About 8 million people are probably infected worldwide， especially in Latin America， where CD is a significant health and socioeconomic problem ［2］. Moreover， CD is becoming increasing widespread in the southern area of the United States，overlapping with the poorest states ［3， 4］. Recently， non-vectorial T. cruzi infection has been increasingly recognized outside endemic areas. Europe， the United States， Canada， Australia， New Zealand and Japan host millions of at-risk Latin American immigrants ［5］. Therefore， the potential of CD becoming a public health issue in that area is considerably high， mainly due to the high number of Latin American immigrants and international travellers， which may contribute for indirect transmission such as blood transfusion， organ transplantation and congenital route； and the presence of potential vectors， triatominae， in this region ［5， 6］.

The available drugs for CD treatment are the 5-nitrofuran，nifurtimox and the 2-nitroimidazole， benznidazole （BZ）. Both drugs present low percentage cure rate， mainly in the chronic phase of the disease， when the majority of cases are diagnosed ［7］. Moreover，available chemotherapeutics are highly toxic， with severe systemic side effects ［7］. The problems associated with the available drugs highlight the urgent need to develop new strategies for chemotherapy against Chagas disease.

Nature has provided an important number of compounds with anti-parasitic activity. For example， quinine， first isolated from the bark of the cinchona tree （Cinchona sp.） was the first effective Western treatment for malaria caused by Plasmodium falciparum，while artemisinin from Artemisia annua L. is still used in malaria treatment in artemisinin-combination therapies. Noteworthy is the fact that the Nobel Prize of Physiology and Medicine 2015 was awarded to three scientists for their discoveries and development of effective drugs against parasitic infections， namely avermectin isolated from Streptomyces avermitilis （and its derivative ivermectin）and artemisinin from Artemisia annua L. （Asteraceae） ［8］. This award emphasizes that nature present unlimited chemical diversity， and highlights the value of natural products as promising alternative therapeutics towards NTDs.

Halophytes are specialized plants able to survive and thrive in saline soils. Although representing only 2% of terrestrial plant species， they are present in about half the higher plant families and have a high diversity of forms. Halophytes have evolved a complex suite of adaptations in response to the osmotic and ionic defies of saline environments that contribute to the generation of reactive oxygen species （ROS）. In order to manage with excessive toxic ROS， halophytes contain antioxidant systems， including enzymes and bioactive compounds， which give them a significant plethora of other biological activities.

Although there are reports of the traditional use of different halophytic species as anti-parasitic and/or anti-helminthic agents［9］，to the best of our knowledge there is no scientific information regarding the potential use of halophytes against NTDs in general，or against CD in particular. Therefore this work evaluated organic extracts made from 31 species of halophytes in vitro against trypomastigotes and intracellular amastigotes forms from Tulahuen strain of T. cruzi. The most active extract was submitted to a bioguided fractionation， and the most promising fraction was chemically characterized by1H-NMR.

2. Material and methods

2.1. Chemicals

All chemicals used in the experiments were of analytical grade， and were purchased from VWR International （Leuven， Belgium）.

2.2. Sample collection

A total of 31 indigenous （Table 1）， mostly obligate， halophyte species were collected from different saline habitats of the southern Portugal （Algarve） at their full flowering time during June of 2013. The researched halophytes belong to 16 plant families and include Aizoaceae （Mesembryanthemum crystallinum L. and Carpobrotus edulis L.）， Amaranthaceae （Arthrocnemum macrostachyum L.，Halopeplis amplexicaulis （Vahl） Ung.-Sternb. ex Ces.， Pass. & Gibelli，Salicornia ramosissima J. Woods， S. fragilis P.W.Ball & Tutin， Salsola vermiculata L.， Sarcocornia perennis （Mill.） A.J. Scott subsp alpini（Lag.） Castrov. and Sarcocornia perennis （Mill.） subsp perennis），Anacardiaceae （Pistacia lentiscus L.）， Asteraceae （Aster tripolium L. and Inula crithmoides L.）， Caryophyllaceae （Spergularia rubra（L.） J.Presl & C. Presl）， Convolvulaceae ［Calystegia （Convulvulus）soldanela （L.） R. Br.］， Cyperaceae （Claudium mariscus （L.） Pohl），F(xiàn)rankeniaceae （Frankenia pulverulenta L. and Frankenia laevis L.）， Gentianaceae （Centaurium erythraea Rafn）， Juncaceae ［Juncus acutus （J. acutus） L.， Juncus inflexus L. and Juncus maritimus Lam.），Lythraceae （Lythrum salicaria L.）， Plumbaginaceae （Limoniastrum monopetalum （L.） Boiss.， Limonium algarvense Erben and Limonium lanceolatum Hoffmanns. & Link）， Polygonaceae （Polygonum maritimum L.） Poaceae （Panicum repens L.， Puccinellia maritima（Huds.） Parl.， Spartina versicolor Fabre）， Tamaricaceae （Tamarix africana Poir） and Typhaceae （Typha domingensis Pers）.

The taxonomical classification was determined by the botanist Dr Manuel J. Pinto （National Museum of Natural History， University of Lisbon， Botanical Garden， Portugal） and voucher specimens are kept in the herbarium of the MarBiotech laboratory （MBH01-MBH31）. Different organs were collected for each species， whenever possible （Table 1）. Plant material was oven dried for 3 days at 40 ℃，powdered and stored at -20 ℃ until needed.

2.3. Preparation of the extracts

Dried samples were mixed with 80% aqueous acetone，dichloromethane and methanol （1：10， w/v） （Table 1）， and extracted overnight at room temperature （RT）， under stirring. Extracts were filtered （Whatman no 4） and concentrated under reduced pressure and temperature （＜ 40 ℃）. Dried extracts were dissolved in dimethyl sulfoxide （DMSO） and stored at 4 ℃ at the concentration of 25 mg/mL until analysis.

2.4. Evaluation of in vitro antitrypanossomal activity，cellular toxicity and selectivity

The in vitro antitrypanossomal activity was evaluated on L929 cells（mouse fibroblasts） infected with the Tulahuen strain of the parasite expressing the Escherichia coli β-galactosidase as reporter gene，according to the method described previously ［10］. The extracts were tested at the concentration of 20 μg/mL， for a period of incubation of 96h. Fractions obtained from the active extract were tested at concentrations ranging from 10 μg/mL to 100 μg/mL， also during a 96h period. Controls with uninfected cells， untreated infected cells，infected cells treated with BZ at the concentration of 1 μg/mL （3.8 μM， positive control） or DMSO （1%， v/v） were used. The results were expressed as the percentage of T. cruzi growth inhibition in extracts-tested infected cells as compared to the untreated infectedcells. Active fractions were evaluated for cytotoxicity and selectivity on uninfected fibroblasts ［10］. Results obtained with the fractions were expressed as IC50values， calculated by linear interpolation and the selectivity index （SI） was determined based on the ratio of the IC50value in the host cell divided by the IC50value of the parasite.

Table 1Botanical names， families， plant parts and extracts used of the 31 halophytic species included in this study.

2.5. Sample fractionation

The active dichloromethane crude extract of J. acutus roots was dissolved in dichloromethane at the concentration of 100 mg/mL，and fractionated by preparative HPLC-DAD （Knauer Smartline，Germany） constituted by the following modules： vacuum degasser（E4320V2）， quaternary pump （EA4300V1） and the diode array detector （E4350）， using a semi-preparative. Analyses were performed on a Luna 5u C18 （2） 100A， （250×10） mm， 5 μm particle size（Phenomenex， Spain）. The mobile phase consisted on acetonitrile（solvent A） and mili-Q water （solvent B） with the following gradient：0-40 min： 80%-10% A， 40-45 min： 10%-0% A， 45-55 min： 0%-0% A， 55-60 min： 0%-80% A， using a flow of 3.5 mL/min. The injection volume was 200 μL and the detector was set at 216 nm. 11 fractions were collected for re-testing for anti-parasitic activity.

2.6. Spectral analysis

NMR spectra were acquired on a Bruker-600 DRX （1H NMR：600 MHz，13C NMR： 150 MHz） spectrometer equipped with a cryo probe， in CDCl3（internal standard， for 1H： CHCl3at δ7.26 ppm；for13C： CDCl3at δ77.0 ppm）.

2.7. Statistical analyses

Results were expressed as Mean ± Standard deviation （SD）， of at least three replicates. Significant differences were assessed by analysis of variance （ANOVA） or using Kruskal-Wallis test （P＜0.05） when parametricity of data did not prevail （SPSS statistical package for Windows， release 15.0）. The IC50values were calculated with GraphPad Prism V 5.0.

3. Results

From 94 samples tested， only was active， namely the dichloromethane extract of J. acutus with an IC50value lower than20 μg/mL. This extract was fractionated by HPLC affording 11 fractions， one of them containing a pure compound （juncunol）which were tested for cytotoxic activity against T. cruzi and mouse fibroblasts （L929 cells）， for the determination of selectivity （Table 2）. From these， fraction 8 was the most active and selective （IC50= 4.1 μg/mL， SI = 1.5）， and was further characterized chemically.

Table 2Effect of the application of fractions from the dichloromethane extract of J. acutus （roots）， on the viability of trypomastigotes and intracellular amastigotes of Tulahuen strain of T. cruzi.

Although fraction 8 gave rather complex1H-NMR spectra a qualitative analysis of the overall spectrum was possible （Figure 1）. The spectrum show typical signals of phenanthrenes （Phe）， 9，10-dihydrophenanthrenes （dPhe） and benzocoumarins （Benz）， all isolated previously from J. acutus （Figure 2， 3） ［11-15］. Peaks have been assigned by comparison with previously published data［10-14］. In the downfield region， between 9.1 and 8.3 ppm， the doublet signals of H-4 proton from phenanthrenes and benzocoumarins are detected. The region between 8.0 and 6.2 ppm shows all the other aromatic signals and H-12 proton from compounds with vinyl chain at C-5. Between 5.9 and 4.7 ppm H-13 protons of vinyl chain（doubledoublets） and carbinol protons of 1-hydroxyethyl （quartet）or hydroxylmethylene （singlet） are observed. Lastly， in the upfield region between 3.2 and 0.70 ppm singlet methyls and multiplets of H-9 and H-10 methylenes of 9，10 dyhydrophenanthrenes are present. Trying to identify some components of the mixture， a 2D NMR experiments were performed （COSY and HMBC）. The analysis of1H-1H COSY evidences， in the downfield region， correlations of doublet at 9.06δ with signal at 7.08δ， doublet at 9.02δ with signal at 7.20δ doublet at 8.99δ with signal at 7.06 δ， and doublet at 8.98δ with signal at 6.48δ. The first three spin systems were assigned at phenanthrenes ［12， 15］， while the last was attributed to a benzocoumarin ［13］. The presence in the 1H NMR of a singlet atδ 3.87 （a methoxyl group）， correlation observed in COSY （7.63 with 6.73δ， 6.80 with 5.54 and 5.25δ）， and methyl singlets at 2.30 and 2.24δ could be attributed at 8-hydroxy-2-methoxy-1，6-dimethyl-5-vinyl-9，10-dihydrophenanthrene ［12］. The presence of this metabolite was confirmed by the long-range heterocorrelations observed in the HMBC spectrum， in fact methoxyl and doublet proton （7.63δ） gave crosspeak with carbon at 156.0 ppm assigned to C-2. Furthermore， a careful analysis of COSY spectrum evidenced a proton 4.15δ （dd，J= 9.4， 7.8 Hz） correlated to 3.19 and 1.91δ， and 5.81δ with 4.98 and 4.44 （dd， J= 17.8， 1.3 Hz）， these two spin system are reliable with a dimeric phenanthrenoid （Figure 3） ［11］. Determinant longrange heterocorrelations of both signal at 4.44 and 4.15 ppm with carbon at 62.0δ， observed in the HMBC spectrum， supported the identification of these metabolites.

Figure 1.1H NMR of J. acutus active fraction.

Figure 2. Structure of phenanthrene， benzocoumarin and 9，10-dihydrophenanthrene.

Figure 3. Structure of dimeric 9，10-dihydrophenanthrene.

4. Discussion

There are several reports on the antiparasitic activity of halophyte species. For example， seed kernels of Caesalpinia crista （Fabaceae）are used in traditional medicine for the treatment of malaria ［16］. That activity was confirmed by in vivo studies using mice infected with Plasmodium berghei ［17］ and was attributed to the presence of cassane- and norcassane-type diterpenes ［18］. The whole aerial organs and roots of Inula cappa （Asteraceae） are also traditionally used for the treatment of malaria ［19， 20］， although the molecules responsible for that activity were not described yet. To the best of our knowledge the antiprotozoal potential of marine halophytes still remains unexplored， especially as possible candidate against CD. In this context， this work evaluated for the first time the anti-T. cruzi activity of organic extracts made from 31 halophyte species abundant in the southern area of Portugal.

Only one extract was able to decrease the growth of parasites，namely the dichloromethane extract of J. acutus roots. Juncus is the largest genus in the Juncaceae family comprising more than 200 species that usually grow in maritime environments. Several Juncus species have medicinal properties e.g. the medulla of J. effusus is used as antipyretic and sedative in Japan and China ［21］. Also，they are used in traditional medicine for the treatment of different health problems. For example， the rhizomes of J. acutus are used for insomnia and the seed of Juncus species for the treatment of stomach disorders ［21］.

To assess which extract components could be responsible for the antiprotozoal activity， the active extract from J. acutus was fractionated by HPLC， affording 11 fractions， one of them containing a pure compound （juncunol）， which were also evaluated for antitrypanossomal activity. When applied at the concentration of 67.7 μg/mL juncunol was able to reduce the parasites growth by 50%. However， it was also cytotoxic against mouse fibroblasts L929 cells. Juncunol is a dihydrophenanthrene previously isolated from of different Juncus species， including J. acutus and J. roemerianus ［15， 22-25］，and has cytotoxic activity towards the microalga Selenastrum capricornutum ［15］， and for several mammalian tumour cell lines ［22］. However， there were no reports until now on its antiparasitic activity. Fraction 8 had the highest activity towards T. cruzi， and contained phenanthrenes， dihydrophenanthrenes and benzocoumarins， which were identified by1H-NMR spectra analysis. Species belonging to the Juncus genus are one of the most prolific sources of phenanthrenes ［18］， which have several biological activities， including antiproliferative， antioxidant， antimicrobial and cytotoxic ［22， 26］. Phenanthrenoids and benzocoumarins obtained from the rhizome of J. acutus have in vitro phytotoxicity， antialgal and anti-inflammatory activities ［12， 14， 15， 27］. Recently， phenanthrene and phenanthrenoids，obtained through a bioguided fractionation of the ethanol extract of J. effuses had in vitro cytotoxic properties towards different cancer cell lines ［28］. Also， a number of benzocoumarins are described with cytotoxic activity against different cells［29］. Moreover，some phenanthrene-derived molecules and benzocoumarins also have anti-parasitic activity. The best example is halofantrine，a phenantherene methanol derivative used in the treatment of malaria ［30］. However， this molecule is no longer recommended in current therapies due to its cardiotoxicity ［30， 31］. More recently，other phenanthrene-based derivatives， particularly 3-hydroxy-N'-arylidenepropanehydrazonamides， have shown potent antimalarial in vitro activities with high selectivity indexes ［32］. Different benzocoumarin scaffolds are highly toxic towards several parasites，including the protozoans Plasmodium spp and Babesia spp［29］. Since phenanthrene-derived compounds and benzocoumarins have previously demonstrated its efficacy as antiparasitic agents， our results strongly suggest that the molecules identified in the active fraction from J. acutus are responsible for its antitrypanossomal activity.

To the best of our knowledge， this is the first report of the potential antitrypanossoma activity of halophyte plants in general， and of the antitrypanosomal activity of J. acutus extract and fractions as well as the isolated compound， juncunol. Based on our results， it is likely that the molecules identified in the active fraction from J. acutus are responsible for its anti T. cruzi activity encouraging further research. In this sense studies aiming the isolation of the bioactive（s） compound （s） of this fraction are already in progress. Moreover，structure-activity relationship （SAR） studies may also disclose a renewed interest in the pharmacological applications of these molecules， by increasing its selectivity towards parasites.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgements

This work was supported by the XtremeBio （PTDC/MAREST/4346/2012） and MaNaCruzi projects （bilateral project， FCT/ CAPES 2358， 2014/2015） funded by FCT - Foundation for Science and Technology and Portuguese National Budget； it also received national funds through FCT project CCMAR/Multi/04326/2013 and P3D-Programa de Descoberta e Desenvolvimento de Drogas（PROEP/CNPq/FIOCRUZ 401988/2012-0）. The authors thank the Program for Technological Development of Tools for Health-PDTISFiocruz for use of its facilities. AJR and SMFM are CNPq Research Fellows. Luísa Custódio was supported by FCT Investigator Programme （IF/00049/2012） and Policarpo Sales by Programa Brasil Sem Miséria / Coordena??o de Aperfei?oamento de Pessoal de Ensino Superior （CAPES） / FIOCRUZ.

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10.1016/j.apjtm.2016.06.015

?Corresponding author： Luísa Custódio， Center of Marine Sciences， University of Algarve， Faculty of Sciences and Technology， Ed. 7， Campus of Gambelas， Faro，Portugal.

Tel.：+351 289 800900 ext. 7381

Fax： +351 289800051

E-mail： lcustodio@ualg.pt.

This work was supported by the XtremeBio （PTDC/MAR-EST/4346/2012） and MaNaCruzi projects （bilateral project， FCT/CAPES 2358， 2014/2015） funded by FCT - Foundation for Science and Technology and Portuguese National Budget； it also received national funds through FCT project CCMAR/Multi/04326/2013 and P3DPrograma de Descoberta e Desenvolvimento de Drogas （PROEP/CNPq/FIOCRUZ 401988/2012-0）. The authors thank the Program for Technological Development of Tools for Health-PDTIS-Fiocruz for use of its facilities. AJR and SMFM are CNPq Research Fellows. Luísa Custódio was supported by FCT Investigator Programme（IF/00049/2012） and Policarpo Sales by Programa Brasil Sem Miséria / Coordena??o de Aperfei?oamento de Pessoal de Ensino Superior （CAPES） / FIOCRUZ.