Biological screening of extracts of Brazilian Asteraceae plants

1 Departamento de Química, Instituto de Ciências Exatas e Biológicas, Universidade Federal de Ouro Preto, Campus Universitário Morro do Cruzeiro, Bauxita, CEP 35400-000, Ouro Preto, MG, Brazil. 2 Departamento de Ciências Biológicas, Campus Senador Helvídio Nunes de Barros, Universidade Federal do Piauí, Rua Cícero Duarte 905, Bairro Junco, CEP 64607-670, Picos, PI, Brazil. 3 Programa de Pós-Graduação em Ciências Farmacêuticas, Núcleo de Tecnologia Farmacêutica, Universidade Federal do Piauí, Ininga, CEP 64.049-550, Teresina, PI, Brazil. 4 Departamento de Fisiologia e Farmacologia, Faculdade de Medicina, Universidade Federal do Ceará, CEP 60430-270, Fortaleza, CE, Brazil. 5 Departamento de Parasitologia, Universidade Federal de Minas Gerais, CEP 31270-901, Belo Horizonte, MG, Brazil.


INTRODUCTION
Currently, there still persists many difficulties and challenges in cancer therapy such as drug resistance, toxicity and low specificity of drugs (Mesquita et al., 2009) (Patel et al., 2009). Over 50% of drugs used in clinical trials for anticancer activity were isolated from natural sources or are related to them (Majumdar, 2012). The three anaerobic protozoa, Entamoeba histolytica, Giardia lamblia and Trichomonas vaginalis are highly prevalent *Corresponding author. E-mail: andnascimen@yahoo.com.br. Tel: +55-31-3559-1769. human-infective parasites with a worldwide prevalence (Cantillo-Ciau et al., 2010). The most effective and commonly used drug in the treatment of these three protozoans is metronidazole. However, this substance has unpleasant side effects such as a metallic taste, headache, dry mouth, urticaria, pruritus, and dark-colored urine (Pérez et al., 2012). Due to these undesired side effects and taking into account the possibility of the development of resistant strains of the T. vaginalis, E. histolytica, and G. lamblia against metronidazole, there is a clear need for new, effective, and safer antiprotozoal agents.
Natural products, especially of plant origin, represent an excellent starting point for research. In traditional medicine there are also several plants used to treat vaginitis (Girón et al., 1988) and amoebic dysentery (Bautista et al., 2011). Amaral et al. (2006) described 153 plant species from 69 families that were evaluated for their giardicidal activity. It was found that the majority of extracts and fractions obtained from plant species employed in popular medicine for the treatment of diarrhea and dysentery exhibited in vitro giardicidal activity, and these were mainly from species belonging to the Asteraceae family.
Asteraceae is the largest family of angiosperms and it comprises 1535 genera and about 23 thousand species distributed in 3 subfamilies and 17 tribes (Bremer, 1994). The plants of the Asteraceae family are very common in the open formations of Brazil, mainly in the cerrado, where the family is well represented by approximately 250 genera and 2000 species . Asteraceae species have been used in the Brazilian folk medicine for several therapeutic purposes. For example, species of the genus Lychnophora, popularly known as "arnica", are widely used in Brazilian folk medicine as anti-inflammatory, to treat bruise, pain, rheumatism and for insect bites (Ferrari et al., 2012). Species of the genus Mikania, known as "guaco", they are widely used in Brazil in the formulation of syrups for the treatment of the respiratory system . Among the native plants of Brazil, species of genus Baccharis, popularly known as "carqueja", has been used as diuretic, tonic, digestive, protective and stimulate of the liver, antianemic, anti-rheumatic, obesity control, diabetes, hepatitis and gastroenteritis (Morais and Castanha, 2011).
Aiming to explore the rich Brazilian biodiversity, we initiated a bioprospection of plants from the Asteraceae family occurring in the state of Minas Gerais, Brazil, by screening plant extracts for cytotoxic and antiprotozoal activities.

Plant material
Seven plants belonging to the Asteraceae family were collected in Ouro Preto-MG, Brazil (April 2010 to April 2012), and were identi-fied by comparison with voucher specimens present in the herbarium, previously identified. Voucher specimens for each plant collected were deposited at the Herbarium José Badini, Universidade Federal de Ouro Preto-UFOP (Table 1).

Extract preparation
Approximately 4 g of the powdered aerial plant material of each specimen was extracted at room temperature by maceration with hexane (100 ml, 3 consecutive extractions over 24 h) followed by extraction using ethyl acetate (100 ml, 3 consecutive extractions over 24 h) and ethanol (100 ml, 3 consecutive extractions over 24 h). The colored solution from each of the plant material was filtered and finally concentrated by vacuum evaporation. The concentrated extract obtained was preserved for further use.
The vials were incubated for 48 h at 37°C. All assays were performed in triplicate and repeated twice. Three vials were used as negative control (inoculum + medium) and three as positive control (Metronidazole, Sigma-Aldrich®). Protozoans viability was qualitatively measured using an inverted microscope (Nikon TMS), to observe trophozoites motility and adherence by comparing with the positive and negative controls.

Hemolytic test
The hemolytic test was performed in 96-well plates following the method described by Berlinck et al. (1996). Each well received 50 ml of 0.85% NaCl solution containing 10 mM CaCl2. The first well was the negative control that contained only the vehicle (1% DMSO), and in the second well 50 ml of test substance that was diluted in half was added. The extracts were tested at concentrations ranging from 1.56 a 200 µM. The last well received 50 ml of 0.2% triton X-100 (in 0.85% saline) to obtain 100% hemolysis. Then, each well received 50 ml of a 2% suspension of mouse or human erythrocytes in 0.85% saline containing 10 mM CaCl2. After incubation at room temperature for 1 h, and centrifugation, the supernatant was removed and the liberated hemoglobin was measured spectroscopically as absorbance at 540 nm.

Phytochemical screening
Chemical tests were carried on the most active extracts (that exhibited high in vitro cytotoxic activities, cell growth inhibition between 75 to 100%) to identify the phytoconstituents, that is, alkaloids, flavonoids, saponins, tannins, and terpenoids, as per the standard procedure ( Table 2.

Statistical analysis
The analysis of cell proliferation (in vitro cytotoxic assays) and hemolytic potential were determined by non-linear regression using the Graphpad program (Intuitive Software for Science, San Diego, CA). Table 1 summarizes the cytotoxic activities displayed by the Asteraceae plant extracts that were evaluated in this research. Of the 21 extracts tested, the analyses by MTT assay showed that 16 (76%) displayed moderate to high in vitro cytotoxic activities against human cancer cells. Ethyl acetate extracts were the most active against the 3

DISCUSSION
The 16 extracts that showed moderate to high in vitro cytotoxic activities against human cancer cells were considered promising anticancer compound sources.
Researches for antineoplasic compounds have demonstrated the great pharmacological relevance of the plant extracts (Ferreira et al., 2011). According to the American National Cancer Institute, the limit to be considered a promising crude extract for further purification is a value lower than 50 μg/ml and cell proliferation inhibition is higher than 90% (Suffness and Pezzuto, 1990;Ferreira et al., 2011). In relation to the cytotoxic or antitumor activity of these plant species, rare findings are available. For example, Acanthospermum australe extracts were capable to increase the survival of Ehrlich ascites tumorbearing mice and stimulated myelepoiesis, which can influence on antitumor immune responses (Mirandola et al., 2002). Studies indicate that some plant substances like polyphenols, epicatechins, steryl glycosides and triterpenoid saponins cause damage to red cell membranes and produce hemolysis (Costa-Lotufo et al., 2002). The mechanical stability of erythrocyte membrane a good indicator of insults by vegetal substances (Sharma and Sharma, 2001;Santos et al., 2010). Then, hemolysis detection is an useful and cheap technique which can displays the effect of increasing concentrations and can to be sigmoidally related to the logarithm contact time, emphasizing the membrane stability as a biological complex to maintain its structure under stress conditions, such as oxidation, hipotonicity, pH changes, heat and in presence of osmotic active solutes (Van Ginkel and Sevanian, 1994;Sharma and Sharma, 2001;Freitas et al., 2008). Herein, none of the extracts tested caused hemolysis even at the highest concentration (200 μg/ml), suggesting that the mechanism of cytotoxicity is probably related to a more specific pathway. Guo and Gao (2013) described the antiproliferative effects of SPV (total saponin extract from Patrinia villosa) and FPV (total flavonoid extract from P. villosa) on four cancer cell lines and concluded that the mechanisms involved in cancer chemoprevention by FPV and SPV extracts were cell cycle arrest and induction of apoptosis. Targeting cell cycle and apoptotic pathways has emerged as an attractive approach for treatment of cancer (Aslantürk and Çelik, 2013).
To our knowledge, no further research was carried out with the species S. urticifolia, M. racemosa, Mutisia campanulata Less, and C. fruticosa.
In this study, the evaluation of the extracts (hexane, ethyl acetate and ethanol) against E. histolytica, T. vaginalis and G. lamblia, was carried out. However, none of the extracts showed antiprotozoal activity.
The Asteraceae plant species tested showed important activity against human tumor cell lines examined. These findings are the base for further studies to isolate (guided by biological assays) and elucidate, the structure of the bioactive compounds assessed from these plants.
Our results are a contribution to a better understanding of the Brazilian biodiversity, which indicate that these natural sources may become an important source for therapeutic agents.