Antiprotozoal activity and cytotoxicity of extracts from Solanum arboreum and S. ovalifolium (Solanaceae)

1 Chemistry of Colombian Plants, Institute of Chemistry, School of Natural and Exact Sciencies, Colombia. 2 PECET, Medical Research Institute, School of Medicine, University of Antioquia, UdeA, Colombia. 3 Group of Botanical Studies, Institute of Biology, Natural and Exact Sciencies Faculty, University of Antioquia UdeA, Calle 70 No. 52–21, A.A 1226, Medellín, Colombia. 4 University of Antioquia Herbarium, Colombia.


INTRODUCTION
Protozoal diseases are a cause of mortality in various developing countries of tropical and subtropical regions.These diseases are significant health problems in endemic countries, this situation is aggravated by increasing treatment failures with available drugs (Bhutta et al., 2014).Leishmaniasis involves a wide spectrum of clinical manifestations, ranging from small cutaneous nodules, plaques or ulcers (cutaneous leishmaniasis) to severe mucosal tissue destruction (mucosal leishmaniasis) or disfunction of vital organs and tissues such as liver, spleen and bone marrow (visceral leishmaniasis).This disease affects more than 12 million people worldwide and is caused by various species of the Leishmania genus, a protozoan parasite that is transmitted to humans through the bite of phlebotominae sandflies of the Lutzomyia genus (Alvar et al., 2012).Chagas disease (also named American trypanosomiasis) affects about 10 million people mainly in Latin America.The disease is produced by the protozoan parasite Trypanosoma cruzi that is transmitted to the mammalian host through the bite of triatomine bugs belonging to Triatoma, Rhodnius and Panstrongylus genus (Nouvellet et al., 2015).On the other hand, malaria is a protozoal disease which affects more than 106 countries, affecting 200 million people and causing about one million deaths annually (Bhutta et al., 2014).Human malaria is caused by at least five species of Plasmodium, the most important being Plasmodium falciparum and Plasmodium vivax (World Health Organization (WHO), 2014).
Current chemotherapies are still based on old drugs; pentavalent antimonials (meglumine antimoniate and sodium stibogluconate) to treat cutaneous leishmaniasis; nitroaromatic compounds (benznidazole and nifurtimox) for treatment of Chagas disease or chloroquine, amodiaquine, sulfadoxine/pyrimethamine to treat P. falciparum or P. vivax malaria, respectively; more recently new Artemisinin-based combination therapy is recommended for the treatment of P. falciparum malaria.Unfortunatelly, all of these drugs have several toxic effects on the patients that are associated with high doses and length of therapeutic schemes.Moreover, they are no longer as effective as before due to the emergence of drug resistance in the parasite, which complicates the problem (Chatelain and Ioset, 2011;Den Boer et al., 2011;Keenan and Chaplin, 2015;Fidock et al., 2004).
Medicinal plant species constitute viable alternatives to conventional medicine in a large number of developing countries, especially poor communities that inhabit rural areas lacking access to health services.S. arboreum Dunal is a shrub of middle size, unarmed, with obovate leaves, 13 to 40 cm long, glabrous beneath.Inflorescenses are short congested cymes, flowers 4 to 8 mm long, 5-merous, white.Fruits globose berries, 1 cm diameter, green and brown when mature.This is a common species, occurring in most forest from Mexico to Peru. S. ovalifolium is a tree, up to 8 m height and 20 cm diameter at breast height (DBH), armed, leaves ovate to lanceolate, pubescent.Inflorescence paniculate, flowers less than 1.5 cm length, sepals green, petals white to lilac.Fruits are green berries.This is an abundant species growing from Venezuela to Peru (Alzate et al., 2012).Habitants of Tumaco (Nariño), on the Colombian Pacific coast, commonly use S. nudum Dunal (Solanaceae) to treat fevers and although leaves extracts of this plant have shown in vitro antimalarial activity against asexual blood forms of P. falciparum (Cardona, 1997;Saez et al., 1998), there are no reports of antiprotozoal activity of the extracts from leaves of S. ovalifolium and S. arboreum.Although steroids of Solanum species are important for their cytotoxicity, it is also known that this cytotoxicity depends on the cell type.
Based on the antiprotozoal activity showed by S. nundum and the importance of evaluating other Solanum species, this study was aimed to evaluate the leishmanicidal, tripanocidal, anti-plasmodial and cytotoxic activity of extracts from S. ovalifolium and S. arboreum, with the purpose of contributing to new therapeutic alternatives that could be used against these protozoal diseases.

Plants
Specimens of S. ovalifolium and S. arboreum were collected during August and September, 2013, respectively, in Santa Elena (Medellín) and Amalfi, respectively, two municipalities of Antioquia department, Colombia.Leaves were collected for chemical and biological studies.Specimens were identified by F. Alzate (Biology institute, University of Antioquia, Medellin, Colombia), voucher specimens are kept at the University of Antioquia Herbarium (HUA) under inclusion numbers 165079 and 183148.

Phytochemical screening
The phytochemical composition of different extracts from S. arboreum and S. ovalifolium was undertaken.In order to detect the presence of steroids, triterpenoids, phenolics, flavonoids, alkaloids, saponins, anthraquinones and anthocyanosids the method described by Yusuf was adopted (Yusuf, 2014).Coumarins was detected by applying the methods of Matos (Matos, 1997).

Biological activity assays
The extracts were subjected to in vitro evaluation of cytotoxicity on U-937 human cells, antileishmanial and antitrypanosomal activities on intracellular amastigotes of L. (V) panamensis and T. cruzi, respectively, and antiplasmodial activity on asynchronous cultures of P. falciparum.

In vitro cytotoxicity
The cytotoxic activity of the extracts was assessed based on the viability of the human promonocytic cell line U-937 (ATCC CRL-1593.2TM ) evaluated by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay following the methodology described previously (Pulido et al., 2012).Briefly, cells grown in tissue flasks were harvested and washed with phosphate buffered saline (PBS) by centrifuguing.Cells were counted and adjusted at 1 × 10 6 cells/ml of RPMI-1640 supplemented with complete 10% fetal bovine serum (FBS) and 1% antibiotics (100 U/ml penicillin and 0.1 mg/ml streptomycin).One hundred µl were dispensed into each well of a 96-well cell-culture plate and then 100 ml of RPMI-1640 and the corresponding concentrations of the extracts were added, starting at 200 µg/ml in duplicate.Plates were incubated at 37°C, 5% CO2 during 72 h in the presence of extracts.The effect of extracts was determined by measuring the activity of the mitochondrial dehydrogenase by adding 10 µl/well of MTT solution (0.5 mg/ml) and incubation at 37°C for 3 h.The reaction was stopped by adding 100 µl/well of 50% isopropanol solution with 10% sodium dodecyl sulfate and 30 min incubation.Cell viability was determined based on the quantity of formazan produced according to the intensity of color (absorbance) registered as optical densities (OD) obtained at 570 nm in a spectrophotometer (Varioskan™ Flash Multimode Reader -Thermo Scientific, USA).Cells cultured in the absence of extracts were used as control of viability (negative control), while meglumine antimoniate (Sbv) and amphotericin B (AmB) were used as control for cytotoxicity (noncytotoxic and cytotoxic drugs, respectively).Assays were performed in two independent runs with three replicates per concentration tested.

In vitro hemolytic activity
The ability to induce hemolysis was evaluated only on those extracts that showed antiplasmodial activity.Briefly, 500 μl of human red blood cells (huRBC), adjusted to 5% hematocrit in RPMI-1640 medium, were placed into each well of a 24-well plate and exposed to four concentrations of each extract (200, 50, 12.5 and 3.125 μg/ml).After 48 h of incubation at 37°C, the concentration of free hemoglobin (as evidence of hemolysis) was determined spectroscopically according to the OD obtained at 542 nm (Varioskan™ Flash Multimode Reader -Thermo Scientific, USA).Non-specific absorbance was corrected by subtracting absorbance of the blank.Determinations were done by triplicate in at least two independent experiments (Conceição et al., 2006).

In vitro antileishmanial activity
The activity of extracts was evaluated on intracellular amastigotes of L. (V) panamensis transfected with the green fluorescent protein gene (MHOM/CO/87/UA140pIR-GFP) (Taylor et al., 2011).Effect of each extract was determined according to the inhibition of the infection evidenced by both decrease of the infected cells and decrease of intracellular parasite load.Briefly, U-937 human cells at a concentration of 3 × 10 5 cells/ml in RPMI 1640 and 0.1 μg/ml of phorbol-12-myristate-13-acetate (PMA) were dispensed into each well of a 24-well cell culture plate and then infected with 5 days-old promastigotes in a 15:1 parasites per cell ratio.Plates were incubated at 34°C, 5% CO2 during 3 h and cells were washed two times with PBS to eliminate non internalized parasites.One ml of fresh RPMI 1640 supplemented with 10% FBS and 1% antibiotics was added into each well, cells were incubated again to guarantee multiplication of intracellular parasites.After 24 h of infection, culture medium was replaced by fresh culture medium containing each extract at 20 μg/ml or lower (based on the cytotoxicity showed previously by each extract) and plates were incubated at 37°C, 5% CO2.After 72 h, inhibition of the infection was determined.For this, cells were removed from the bottom plate with a trypsin/EDTA (250 mg) solution; recovered cells were centrifuged at 1100 rpm during 10 min at 4°C, the supernatant was discarded and cells were washed with 1 ml of cold PBS and centrifuged at 1100 rpm during 10 min at 4°C.The supernatant was discarded and cells were suspended in 500 μl of PBS and analyzed by flow cytometry (FC 500MPL, Cytomics, Brea, CA, US).All determinations for each extract and standard drugs were carried out in triplicate, in two independent experiments (Buckner et al., 1996;Pulido et al., 2012).Activity of tested extracts was carried out in parallel with infection progress in culture medium alone and in culture medium with AmB and Sbv as antileishmanial drugs (positive controls).Extracts that showed percentages of inhibition higher than 50% to 20 or fewer μg/ml were then evaluated at four additional concentrations to determine the effective concentration 50 (EC50).Here, infected cells were exposed against each concentration of extracts during 72 h; then, cells were removed and tested by flow cytometry as described before.

In vitro trypanocidal activity
Extracts were tested on intracellular amastigotes of T. cruzi, Tulahuen strain transfected with β-galactosidase gene (donated by Dr. F. S. Buckner, University of Washington) (Buckner et al., 1996).The activity was determined according to the ability of the extract to reduce the infection of U-937 cells by T. cruzi.Following the procedure previously described, anti-parasite activity was initially screened at a single concentration of 20 mg/ml.In this case, 100 μl of U-937 human cells at a concentration of 2.5 × 10 5 cells/ml in RPMI-1640, 10% FBS (Fetal Bovine Serum) and 0.1 μg/ml of (Phorbol 12-myristate 13-acetate) were placed in each well of 96well plates and then infected with phase growth epimastigotes in 5:1 (parasites per cell) ratio and incubated at 34°C, 5% CO2.After 24 h of incubation, 20 μg/ml of each extract were added to infected cells.After 72 h of incubation, the effect of all extracts on viability of intracellular amastigotes was determined by measuring the βgalactosidase activity by spectrophotometry, adding 100 μM CPRG (Chlorophenol red-β-D-galactopyranoside) and 0.1% nonidet P-40 to each well.After 3 h of incubation, plates were read at 570 nm in a spectrophotometer (Varioskan™ Flash Multimode Reader -Thermo Scientific, USA) and intensity of color (absorbance) was registered as OD.Extracts that showed inhibition percentages higher than 50% were evaluated again at four concentrations selected according to the LC50 previously obtained for each extract.Infected cells exposed to benznidazol (BNZ) were used as control for antitrypanosomal activity (positive control) while infected cells incubated in culture medium alone were used as control for infection (negative control).Non-specific absorbance was corrected by subtracting the OD of the blank.Determinations were done by triplicate in at least two independent experiments (Insuasty et al., 2015).

In vitro Antiplasmodial Activity
The antiplasmodial activity was evaluated in vitro on Londoño et al. 103 asynchronic cultures of P. falciparum (NF54 strain), maintained in standard culture conditions.The effect of each extract over the growth of the parasites was determined by quantifying the parasite DNA stained with ethidium bromide (EtBr) (Insuasty et al., 2015;Insuasty et al., 2013).Briefly, unsyncronized P. falciparum cultures were adjusted to 1.5 to 2% parasitemia and 5% hematocrit in RPMI medium enriched with 10% human serum (complete medium).
Then, in each well of a 96-well plate, 500 μl of parasite suspension were dispensed and subsequently exposed against 500 μl of four concentrations of extracts (200, 50, 12.5 and 3.125 μg/ml).Dilutions were prepared from a stock solution of 1.000 µg/ml.Chloroquine (CQ) was used as positive antiplasmodial drug control.Parasites unexposed to any compound were used as control of both growth and viability (negative control).Plates were incubated for 48 h at 37°C in N2 (90%), CO2 (5%) and O2 (5%) atmosphere.After incubation, parasite DNA was extracted and purified by using a lysis solution containing proteinase K. DNA was stained with EtBr and then quantified in a spectrofluorometer (Varioskan, Thermo) reading at 542 nm.The intensity of fluorescence in each experimental condition was registered in arbitrary units of fluorescence (AUF).Non-specific fluorescence was corrected by subtracting fluorescence of unstained DNA.Determinations were done by triplicate in at least two independent experiments.

Data analysis
Cytotoxicity was determined according to cell growth (viability) and mortality percentages obtained for each isolated experiment (extracts, AmB, Sbv and culture medium alone).Results were expressed as 50 lethal concentrations (LC50), corresponding to the concentration necessary to eliminate 50% of cells, calculated by Probit analysis (Finney, 1978).Percentage of viability was calculated by Equation 1, where the optical density (OD) of control corresponds to 100% of viability (cell growth).
Antileishmanial activity was determined according to percentage of infected cells and parasite load obtained for each experimental condition by flow cytometer.The percentage of infected cells was determined as the number of positive events by double fluorescence (green for parasites and red for cells) using dotplot analysis.On the other hand, the parasitic load was determined by analysis of mean fluorescence intensity (MFI) (Pulido et al., 2012).The parasitemia inhibition was calculated by equation 2, where the MFI of control corresponds to 100% of parasitemia.In turn, inhibition percentage corresponds to 100 -% Parasitemia.Results of antileishmanial activity was expressed as 50% effective concentrations (EC50) determined by the Probit method (Finney, 1978): % Parasitemia = (MFI Exposed parasites) / (MFI Control parasites) × 100 (2) Similarly, trypanocidal activity was determined according to the percentage of infected cells and parasite load obtained for each experimental condition by colorimetry.The parasite inhibition was calculated by equation 3, where the OD of control corresponds to 100% of parasites.In turn, inhibition percentage corresponds to 100 -% of Parasites.Results of trypanocidal activity were also expressed as EC50 determined by the Probit method (Finney, 1978):  Then, the inhibition growing percentage was calculated according to equation 5: The hemolytic activity of tested extracts was evidenced by the percentage of hemoglobin free content calculated according to equation 6: The cytotoxicity was graded according to the LC50 value as high cytotoxicity: LC50 < 100 μg/ml, moderate cytotoxicity: LC50 > 100 to < 200 μg/ml, and potentially non-cytotoxicity: LC50 > 200 μg/ml.Antiprotozoal activity (leishmanicidal, trypanocidal, or antiplasmodial) was graded according to the EC50 or IC50 values as high activity: EC50 < 20 μg/ml, moderate activity: EC50 > 20 to < 50 μg/ml, potentially non activity: EC50 > 100 μg/ml.The selectivity index (SI) was calculated by dividing the cytotoxic activity and the leishmanicidal activity using the following formula: SI = LC50 / EC50 or IC50.

Phytochemical screening
Preliminary phytochemical analysis showed a similar composition of secondary metabolites in both species.This analysis revealed the presence of triterpenes in all extracts, while phenols were only present in ethyl acetate and ethanol extracts.Saponins, flavonoids and anthocyanosids were found in the polar extracts and coumarins were detected in the ethyl acetate extracts, in both species.Coumarins were also observed in the ethanol extract of S. arboreum.No anthraquinones nor alkaloids were found in any extract.The results are listed in Table 1.

Identification of steroids
Comparative studies by thin layer chromatography of main secondary metabolites of Solanum nudum with S. ovalifolium and S. arboreum extracts are shown in Table 2.The presence of compounds 1 and 2 is observed in the hexane and dichloromethane extracts from S. arboreum (H2 and D2, respectively).Diosgenone 5 was detected in dichloromethane extract from S. ovalifolium (D1) and hexane, dichloromethane and ethyl acetate extract (H2, D2 and EA2, respectively) from S. arboreum.

Biological activities
The effect of extracts on cell growth (viability) was assessed in human macrophages (U-937 cells) which are the host cells for L. (V) panamensis and T. cruzi parasites.On the other hand, the antiparasite activity of these extracts was tested on intracellular amastigotes of L. (V.) panamensis and T. cruzi and total forms of P. falciparum according to the ability of extracts to reduce the amount of parasites after exposure.
When protozoal activity was compared with cytotoxicity, we found that biological activity of D2 was selective for L. panamensis (SI 5.1) while D1 and H1 were selective for T. cruzi with SI 2.4, and 1.1, respectively (Table 4).H2 was selective for P. falciparum (SI 1.7), Et2 was selective for T. cruzi and L. panamensis (1.6 and 1.0, respectively).The selectivity of H2 was higher than that showed by CQ (1.7 vs 0.34).Although D2 extract showed better activity than SbV, its SI was affected by high cytotoxicity.Similarly, Et2 showed activity comparable to benznidazole, with the SI also being affected by the high cytotoxicity.
Et2 and D2 extracts of S. arboreum showed activity against all parasites tested here: L. panamensis and T. cruzi and P. falciparum; the activity of the Et2 extract is probably due to the presence of polar compounds such as saponins, flavonoids and coumarins (Table 1), which have long been recognized to exhibit antiprotozoal activity (Pierson et al., 2010;Maes et al., 2004;Robledo et al., 2015;Dos santos et al., 2009).The activity of D2 extract is probably due to the presence of steroids such as diosgenone (Table 2).This situation could also explain the antiplasmodial activity shown by H2 extract from S. ovalifolium.
Althouhgh the action mechanism of steroids is unknown, it is accepted that these compounds may be initiated at the cell membrane (Haines, 2001), but also via intracellular receptor binding (Krauss, 2001).In addition, steroids may participate in growth regulation, proliferation and cell death (Devkota et al., 2007;Freilich et al., 2000;Pabón et al., 2002) and redox mechanisms (Pabón et al., 2009).These compounds could participate in a conjugated addition of nucleophilic amino acid residues present in target enzymes such as cysteine proteases on leishmania (Mottram et al., 2004) in a Michael type mechanism.This mechanism has been reported for other α,β-unsaturated compounds such as lactones and chromones (Cardona et al., 2014;Otero et al., 2014).
Another study of S. arboreum showed that ethanolic extracts of roots and fruits were active against promastigotes of Leishmania sp.(OCR with known characteristics) with IC 50 of 25.8 and 72.5 µg/ml, respectively.Ethanolic extracts of leaves of this plant were not active (Chinchilla et al., 2014).This result is opposite to what we found in this research, which demonstrates the potential antiprotozoal activity of S. arboreum.This result suggests that there is no correlation in activity between promastigotes and amastigotes when it comes to different species of parasites, L. mexicana vs L. panamensis, which differ in the clinical forms and treatment response (WHO, 2010).Another explanation could be related to the fact that contents of most of the chemical constituents varied significantly with diff erent seasons (Hussain et al., 2008), collection place (Djouahri et al., 2015), and altitude (Zidorn et al., 2005).A comparative analysis between the ethanolic extracts of S. arboreum (Et2) tested here (Table 3) and S. nudum (SNMet) evaluated by others (García-Huertas et al., 2013) showed that S. arboreum had better antiplasmodial activity than alcoholic extracts of S. nudum (IC 50 17.3 ± 1.3 vs 54.8 ± 8.1 µg/ml, respectively, data not shown).The poor antiprotozoal activity shown by S. ovalifolium could be due to the low concentration of secondary metabolites present in the extracts.

Conclusion
Leishmanicidal, tripanocidal, anti-plasmodial and cytotoxic screening of eight extracts from two Solanum species are reported.Based on activity observed on intracellular amastigotes of L. panamensis and T. cruzi and total forms of P. falciparum of dichloromethane D2 and ethanol Et2 extracts of S. arboreum, suggests that these extracts may be considered as promising in the search for new antiprotozoal compounds.However, additional studies on toxicity using other cell lines are needed in order to discriminate whether the toxicity shown by these extracts is against tumoral or nontumoral cells.

Table 2 .
Steroids detection in Solanum arboreum and S. ovalifolium extracts by thin layer chromatography.

Table 4 .
Selectivity of biological activity of Solanum arboreum and S. ovalifolium extracts.