The genre Croton comprises more than 1300 species of trees, bushes, and herbs distributes all over the world. Such biodiversity is mainly found in India, Brazil and Madagascar where its ethnomedicinal value is recognized (Yankep et al., 2003; Ye et al., 2012). Croton heliotropiifolius Kunth is an endemic specie in the northeast of Brazil frequently found within “caatinga”, “brejo”, “resting” and “cerrado” vegetations. It is known by unofficial names such as “velame”, “velaminho” and “velame-de-cheiro” due to the presence of trichomes. Previous studies have described the presence of alkaloids, polyphenols and reducing agents in C. heliotropiifolius, including its medicinal use for relief of stomach pain, vomit, diarrhoea and as an antithermic (Randau et al., 2004). The essential oil has been described as larvicidal against Aedes aegypti (Dória et al., 2010) and the ethanolic extract demonstrated significant insecticidal activity against Sitophilus zeamais (Silva et al., 2012). The Human Toxicity Potential (HTP) is an important test to be performed early in the study of a medicinal herb. It is recommended that new herbs and those with unknown actions shall be analysed in a controlled cellular environment as free of complex interactions inherent an organism. The methodologies is aimed at analysing the cellular behaviour, display several advantages such as low costs, easy and quick execution and controlled cell environment (Freshney, 2000). Cancer is a progressive chronic disease responsible for approximately 13% of deaths during last year (WHO, 2016). Genre, race, age, genetic predisposition and environmental exposure to carcinogenic agents, are directly related to the distribution and incidence of tumours (Chu et al., 2004).

General experimental procedures

The methanolic extract of C. heliotropiifolius leaves was concentrated using a rotary evaporator with vacuum BUCHI Switzerland at 60ºC. Thin-layer chromatography (TLC) was performed with TLC Silicagel 60G F254 (Merck®) on top of an aluminium base. Compounds were sprayed with visualization reagents, visualized under UV light (254 nm - 365 nm), and for flavonoids, was compared with the quercetin standard. The HPLC system used was a HPLC Waters (Self Cleaning  System)  attached to an UV spectrophotometer with detector model Waters 2998 PDA containing photodiodes (Photodiode Array Detector). Values in the infrared region (FTIR) and UV- visible spectrum were registered respectively by an Agilent 630 and Shimadzu UV-vis 1800 spectrophotometers. All chemical products used in those procedures were for analytic use (Sigma® e Merck®). The Division of Antibiotics from the Federal University of Pernambuco (UFPE) supplied the cells, antibiotic solutions (penicillin and streptomycin), and the drug doxorubicin. Both culture media, Dulbecco MEM (DMEM) and Roswell Park Memorial Institute (RPMI), and the fetal bovine serum (FBS) were Gibco™ BRL. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was manufactured by Sigma®.

Plant material

C. heliotropiifolius (Euphorbiaceae) leaves were collected in July 2015 in the urban area of Garanhuns city, Pernambuco State, Brazil (Latitude: -8.89074, Longitude: -36.4966 8° 53′ 27″ Sul, 36° 29′ 48″ Oeste). The plant was identified by the Dárdano de Andrade Lima herbarium in the Agronomic Research Institute (IPA). It was registered in the referred herbarium under the catalog number 90440 in which an exsiccatae was deposited.

Extraction of compounds

The extract composed of plant leaves was prepared by soaking and maceration (Cechinel Filho and Yunes, 1998). About 150 g of powdered material was taken in a clean, flat bottomed glass container and soaked in 200 mL of methanol. The container with its contents was sealed and kept for a period of 10 days accompanying occasional shaking and stirring. Next, filtration was performed followed by evaporation of all solvent to obtain the crude extract. The presence of flavonoids, alkaloids and coumarins was tested by thin layer chromatography (TLC). The mobile phase used for flavonoids and alkaloids was ethyl acetate-formic acid-acetic acid-water (AcOEt- HCOOH-AcOH-H2O 100:11:11:27v/v), for coumarins ether-toluene- acetic acid 10% (50:50:50v/v) was used. The visualization reagents used for flavonoids and alkaloids were respectively etilborilaminoester acid (Neu) and Dragendorff. For Coumarins, the visualization method used was UV light on 365 nm (Wagner et al., 1996). The presence of saponins was tested by mechanical shaking of the extract and visualization of foam (Simões et al., 2004). Formation of foam for 15 min was considered as positive for presence of saponins (Dewick, 2002). The presence of tannins was investigated by addition of iron chlorine 0.5 M to the hydrated form of extract. In this method, the formation of blue precipitate indicates the presence of hydrolysed tannins awhile green precipitated the presence of condensed tannins (Matos, 1997). HPLC was conducted in model Shimadzu®HPLC LC-10, XBridge C18 column with 4.6 mm x 250 mm dimensions, 5 µm of particle size and 0.7 mL/min flow. The solvent system displayed; 94% of H₂O and 6% of acetonitrile on 0 min (A), 65% of H₂O and 35% of acetonitrile on 8 min (B), 94% of H₂O and 6% of acetonitrile on 9 min (C); with 40 µL of injection volume and sample concentration of 20 mg/mL in 50% MeOH. Identification was performed using the standard method and external integration of the peaks at 256 nm for gallic acid. The spectroscopic profile of the extract was obtained by Fourier transform infrared spectroscopy (FTIR). In the semisolid state, the extract was pressed against the diamond crystal of the equipment. Next, readings were recorded. The FTIR spectrum was obtained in less than 30 s and results compared against standards samples from the library. An aliquot (10 ml) of the extract (1 mg) diluted in MeOH (100%) was used to obtain the UV-visible spectrum, using the wavelength range of 200 to 700 nm.