In vitro trypanocidal activity of solamargine and extracts from Solanum palinacanthum and Solanum lycocarpum of brazilian cerrado

The present investigation was to evaluate the potential trypanocidal activity of crude ethanolic extract of the fruits of Solanum palinacanthum, Solanum lycocarpum and the glycoalcaloid, solamargine. S. palinacanthum and S. lycocarpum fruit powders were submitted to exhaustively extraction with 96% ethanol and solamargine were isolated from the extract of S. palinacanthum. Both extracts and solamargine were analysed for trypanocidal activity by using MTT colorimetric assay. Extracts of S. palinacanthum showed to be more active (IC50 = 175.9 μg.ml-1) than S. lycocarpum (IC50 = 194.7 μg.ml-1). Solamargine presented a strong activity (IC50 = 15.3 μg.ml-1), which can explain the better activity of the both extracts. Benznidazol (IC50 = 9.0 μg.ml-1) is the only drug used to treat Chagas’ disease. These findings demonstrate for the first time that ethanol extracts obtained from both fruits of S. palinacanthum and S. lycocarpum and also solamargine have a potential anti-trypanosomal activity.


INTRODUCTION
Solanum palinacanthum Dunal.and Solanum lycocarpum St. Hill are members of the Solanaceae family, found in tropical and temperate zones, including the Brazilian Cerrado.They have great importance for food and medicinal use (Blankemeyer et al. 1998, Almeida et al. 2010).The antiviral, diuretic, anti-spasmodic, anti-inflammatory and other properties of these plants have been intensively studied (Balasubramanian et al. 2007; as cited by Soares-Mota et al. 2009).However, attention has not been given for other healing properties.
Phytochemical studies revealed that polyphenols and glycoalcaloids, the most common elements obtained from some species of Solanum, which are secondary synthesized metabolites, seek protection against animals and insects because of [903][904][905][906][907] their toxicity (Hall et al. 2006).Glycoalkaloids solamargine and solasonine were identified in various species of Solanaceae family, which have extensive anti-viral function (Hall et al. 2006, Sun et al. 2010).However, the trypanocidal effects of the glycoalkaloids and plants extracts have never been reported before.The aim of the present investigation is to evaluate the potential trypanocidal activity of 95% crude ethanol extracts of the fruits of S. palinacanthum, S. lycocarpum and solamargine isolated from S. palinacanthum, the major constituent of these plants.

PLANT MATERIAL AND ExTRACTION
The study was performed with fruits from a Solanum palinacanthum Dunal.Fruits powder (500 g) of S. palinacanthum were submitted to exhaustively extraction with 96% ethanol (EtOh) by four times at temperature (4 oC).The solvent proportion corresponded to five times the plant material.Subsequently, exhaustive extraction with hot EtOh was done and evaporation of the solvent under reduced pressure afforded a crude extract (100.2 g).The crude extract was redissolved in 5% aqueous acetic acid (v/v) and after 24 hours, a preliminary purification involving defatting was done by successive benzene extraction.The final solution was changed to ph 10.0 with ammonium hydroxide.After 24 hours at room temperature the precipitated was obtained, filtered and dried (24.2 g).
Fruits powder (35 g) of Solanum lycocarpum was submitted to exhaustively extraction with 250 mL of 96% ethanol by 4 hours at boiling temperature, followed by filtration under reduced pressure.These processes were repeated with 200 mL of 96% ethanol.Were blended the extracts and concentrated under reduced pressure until syrupy consistency and then dried to room temperature by air suction (8.12 g) (Almeida et al. 1995).
ISOLATION ANd IdENTIFICATION OF SOLAMARGINE The glycoalkaloid mixture of Solanum palinacanthum was subjected to chromatographic column (aluminum oxide, neutral) and after elution with 40% aqueous ethanol (v/v) yielded solamargine (0.92 g) as showed in the Fig. 1.The separation was monitored by TLC in solvent system (n-butanol:acetic acid glacial:water (6:3:1), nebulized and developed with 10% aqueous sulfuric acid (v/v) and then heated at 150 °C for 5 min.Solamargine was obtained and rechromatographed on aluminum for purification and repeatedly recrystallised with methanol, and its identity ascertained by comparing physical and spectral data (mp, IV) (Almeida et al. 1995).An in vitro assay was performed measuring the viability of Trypanosoma cruzi (Y strain) epimastigote forms growing in axenic culture using MTT assay (Cotinguiba et al. 2009).The parasites were grown axenically at 28 °C in liverinfusion Tryptose (LIT) medium supplemented with 10% fetal calf serum and harvested during the exponential phase of growth (7 day-old culture forms).Extracts and compounds were dissolved in dimethylsulfoxide (dMSO) and further added to a 96-well tissue culture plate (TPP) in different final concentrations.T. cruzi (1 x 10 7 parasites.mL - ) were added into each well and the same quantity of LIT medium was added into the control wells.These plates were maintained at 28 °C for 72 h, 10 μL of a 2.5 mg.mL -1 MTT-PMS solution was added to each well and the plates were incubated for 75 min in the dark at 28 °C.A solution of 10% (100 μL) of sodium dodecyl sulfate (SdS) was added to the anterior solution and maintained at room temperature and in the dark at 30 min.The absorbance of the samples had read at 595 nm.The 50% inhibitory concentration (IC 50 ) values were determined by linear regression analysis after a 72 h incubation period.All the tests were done in triplicates and the IC 50 values of samples and benznidazole (positive control) were determined.For the statistical analysis, probit's method was employed (Muelas-Serrano et al. 2000).

RESULTS AND DISCUSSION
Chagas' disease or American trypanosomiasis caused by the protozoan flagellate T. cruzi, is an important public health problem in Latin America, affecting millions of people annually (Sanchez-Burgos et al. 2003).The most common treatment for this disease involves two drugs, benznidazole (A) and nifurtimox (B) (Fig. 2), which are active only during the acute and short-term chronic phases.Beznidazole (Rochagan ® /Brazil and Radanil ® /Argentina) is now the only drug still available since the production of nifurtimox was discontinued.Besides developing severe side effects, narrow therapeutic windows, and variable drug susceptibilities among T. cruzi strains, result in low clinical efficacies for these 2-nitroimidazole (Coura and Castro 2002).In many Solanum species, the glycoalkaloids are presented as pairs of structurally related compounds which share a common aglycone with either solasodine and differ only in their carbohydrate moiety (Chataing et al. 1998).The literature contains several reports on the biological activity of nitrogen-containing steroids of the species Solanum (Chataing et al. 1998, Hall et al. 2006).
In this work, we presented the inhibitory activity the ethanol extracts from fruits of S. palinacanthum and S. lycocarpum, as well as the purified solamargine, comparing with benznidazole (as positive control).The results are shown in Table I.
The solamargine is a dominant steroidal glycoalkaloid extract from plants belonging to the genus Solanum and showed an IC 50 very close to the benznidazole.Similar results were showed by other authors in the past (Chataing et al. 1998, Hall et al. 2006, Valaderesa et al. 2009).RAQUEL R.D. MOREIRA et al.
There was a light difference in the response between the two extracts: IC 50 = 175.9µg.ml -1 for extract of S. palinacanthum and IC 50 = 194.7 µg.ml -1 for S. lycocarpum.
Solamargine isolated from S. palinacanthum showed an IC 50 = 15.3 µg.ml -1 , which was very interesting by explaining the better activity of the both extracts.Solamargine had also a pronounced effect on T. cruzi, closely similar to benznidazole (IC 50 = 9.0 µg.ml -1 ).However, other methodologies should be used to rightly understand the mechanism of action.
Our results were very similar to those described by Hall et al. (2006), but using different approach.They have speculated that the positioning of the terminal sugars in α-solamargine is sterically more favorable to bind with the parasites mucin-rich cell surface than those of α-solasonine (hall et al. 2006).Once bound in sufficient concentrations, the alkaloid's lipid moiety would induce cytolysis through hydrophobic interactions with membrane sterols (Hall et al. 2006).
Since the alkaloid used in this study was derived from ripe fruits of a Solanum species.There have been numerous hypotheses put forth to explain the allocation of secondary metabolites in ripe fleshy fruits (hall et al. 2006(hall et al. , Li et al. 2007)), including the retention of high levels of glycoalkaloids in fruits of some Solanum species (Kuo et al. 2000, Hall et al. 2006).
The 96% ethanolic extracts from the fruits of S. palinacanthum and S. lycocarpum exhibited the most potent in vitro trypanocidal activity.These species are an interesting group for more studies and search of novel therapeutic plants against Chagas' disease (Almeida et al. 1995;Soares-Mota et al. 2009).

CONCLUSION
It can be concluded from this study that 96% ethanolic extracts from the fruits of S. palinacanthum and S. lycocarpum presented a potential anti-trypanosomal activity based on toxic effect against epimastigote form of T. cruzi Y strain.The glycoalkaloid solamargine has been isolated from S. palinacanthum and demonstrated to be active in the trypanocidal effect and would be a candidate to new therapeutic substance.
In view of these findings, further chemical and pharmacological investigations to identify others secondary metabolites and to evaluate the potential of these Solanum species as antichagasic agents in vivo are recommended.
and Solanum lycocarpum St. Hill collected in Itápolis and Barretos, respectively, cities within districts of São Paulo State, Brazil.The voucher specimen of S. palinacanthum Dunal.

TABLE I In vitro trypanocidal activity of ethanolic extracts of S. palinacanthum and S. lycocarpum and solamargine.
* positive control.