Antiproliferative effect of the hydroalcoholic extract of Hymenaea stigonocarpa Mart . ex Hayne ( Fabaceae , Caesalpinioideae ) on the meristematic cells of Allium cepa L . roots

This study evaluates the hydroalcoholic extract obtained from the cork cambium of Hymenaea stigonocarpa (jatobá-do-cerrado) on the meristematic cells of Allium cepa roots, at 3 concentrations, 0.5, 1.0, and 1.5 mg/ mL, and 2 exposure times, 24 h and 48 h. The slides were made through the crushing technique. Cells were analyzed throughout the cell cycle, totaling 5,000 for each control and concentration group. We found that the 3 concentrations signiicantly decreased the cell division rate at the exposure time of 48 h when compared to their respective controls. The mitotic index of the exposure time of 48 h was statistically different when compared to the cell division rate of the exposure time of 24 h for all concentrations. The number of cell abnormalities observed was not statistically signiicant by means of the chi-square test used. Therefore, under the conditions analyzed, the 3 concentrations tested for the cork cambium of jatobá-do-cerrado signiicantly decreased, in statistical terms, the cell division rate in the meristematic cells of A. cepa roots.


Introduction
Leguminosae is a plant family of great economic and medical importance to temperate and tropical regions worldwide.Among these plants, the species Hymenaea stigonocarpa Mart.ex Hayne, known in Brazil as jatobádo-cerrado, is mainly found in the states of the Central-West, North, and Northeast regions, in open formations of cerrado and caatinga.
The human population in these regions consumes the outer surface of the bark of this legume species in tea form, in order to cure urinary tract infections (DIMECH et al., 2013), alleviate stomach pain, cure intestinal infections (ORSI et al., 2014), ight against helminths (VALENTE et al., 2014), and treat depression (ORSI et al., 2014).According to Cartaxo et al. (2010), this bark contains many groups of metabolites, such as diterpene acids, anthraquinones, high concentrations of minerals, tannins, lavonoids, xyloglucan, and oligosaccharides.
So far, there are no phytochemical studies on Hymenaea stigonocarpa, thus its bioactive compounds are not known.
Although the medicinal properties of the bark of this species have already been scientiically proven in laboratory experiments and despite its wide use by the population in these three Brazilian regions, no data was found in the literature regarding its toxic potential at the systemic and cellular levels.However, according to Cartaxo et al. (2010), studies have shown that the bark of some Hymenaea species exhibited cytotoxic and mutagenic potential in the culture of both normal and tumor cells, as well as by using an in vivo test system.Thus, it is worth analyzing the action of the bark of Hymenaea stigonocarpa at the cell level.
Bioassays with plants are regarded as highly sensitive, quick, and simple to monitor toxic effects of chemical compounds at the cell level.Among them, the meristematic cells of Allium cepa (onion) root are eficient test organisms for the irst screening for cytotoxicity of herbs (SABINI et al., 2011;HERRERO et al., 2012), given their kinetic proliferation properties, as they have few and large chromosomes (2n = 16), something which facilitates their analysis (CARITÁ; MARIN-MORALES, 2008), and they allow good visualization of cell abnormalities when present (BAGATINI et al., 2007).Besides, Fachinetto et al. (2007) report that the results obtained by means of this test system are excellent parameters for cytotoxicity and genotoxicity analysis and they have been used as an indicator to warn the population about the consumption of certain foods and synthetic and natural medicines.
In this context, this study aims to assess the activity of the hydroalcoholic extract from the bark of H. stigonocarpa on the cell cycle of A. cepa roots at different concentrations and exposure times.

Material and Methods
For this study, bark pieces of H. stigonocarpa were purchased from a herbal store in Picos, Piauí, Brazil.The study was carried out in December 2013 and January 2014.
Effect of Hymenaea stigonocarpa Mart.at the cell level

Obtaining the hydroalcoholic extract
Before extraction, the bark pieces were dried at room temperature for 5 days and ground in a knife mill.Then, the plant material was weighed and placed in infusion in a 80% ethanol extraction solution at a 1:5 ratio (COUTINHO; HASHIMAMOTO, 1971) and stored at room temperature for 11 days, shaking the macerate once a day.After this procedure, the material was iltered by using ilter paper and the iltrate was concentrated in a rotary evaporator, under low pressure.

Cytotoxicity test using Allium cepa L.
To assess the cytotoxicity, we established 3 concentrations of the hydroalcoholic extract, 0.5, 1.0, and 1.5 mg/mL.Bulbs of Allium cepa were allowed to root in lasks with distilled water at 25°C, under constant aeration, until obtaining roots about 1.0 cm long.For analyzing each concentration, an experimental group was set with 5 bulbs.Before placing the roots in contact with their respective concentrations, some of them were collected and ixed to serve as control (CO) for the bulb itself.Then, the remaining roots were placed at their respective concentrations for 24 h; this procedure is referred to as the 24 h exposure time (24 ET).
After this period, some roots were removed and ixed.The remaining roots of each bulb were placed in contact with their respective concentrations of extract for further 24 h (48 h exposure time -ET 48).After this, roots were collected and ixed.The material was ixed in 3:1 Carnoy's ixative (ethanol: acetic acid) for about 24 h.On average, 5 slides were prepared per bulb, according to the protocol proposed by Guerra and Souza (2002) and analyzed through light microscopy at 40× objective.For each bulb, 1,000 cells were analyzed, totaling 5,000 cells for each tested concentration of the extract.During the analysis, we observed cells in interphase, prophase, metaphase, anaphase, telophase, and the presence of aneugenic effects and micronuclei.Mean values were calculated for each phase of the cell cycle of A. cepa and the mitotic index (MI) was determined.Statistical analysis of data was performed by means of χ2 test with a probability level < 0.05, using the software BioEstat, version 5.0 (AYRES, 2007).

Results and Discussion
Table 1 lists the number of cells in interphase and at different cell division stages and the mitotic index values obtained for the cells of A. cepa roots treated with water (CO) and with the concentrations of the extract obtained from H. stigonocarpa for 24 h and 48 h.Signiicant X 2 values are also presented.
The results (Table 1) indicated no significant difference between MI of CO and MI of ET 24 for any of the 3 treatments.On the other hand, MI of ET 48 h was signiicantly different from the cell division index of CO and TE 24 h, in the 3 concentrations.
It is worth mentioning that, at all concentrations tested, despite ET 24 h had no reduction in the mitotic index in relation to their respective controls, it may be seen that the number of cells in prophase, when the mitotic spindle has not been organized, yet, is higher than in other cell division stages (Table 1).
Thus, under the conditions studied, the 3 concentrations promoted antiproliferative effect on the meristematic cells of A. cepa root in ET 48.Cell abnormalities were found, such as anaphase and telophase bridges, micronuclei and colchicinemetaphase, in ET 48 for the 3 concentrations, however, the numbers were not signiicant (p > 0.05).Lacerda et al. (2014) investigated the activity at the cell level of the bark aqueous extract of H. stigonocarpa on meristematic cells of A. cepa root at concentrations commonly used in traditional medicine by the population and they found that the three concentrations tested caused statistically signiicant inhibition on the cell cycle of the test system used.Orsi et al. (2014), through classic and gas chromatography, they found that the hydroalcoholic extract of the bark of jatobá-do-cerrado consists of high lavonoid and tannin concentrations.According to Silva et al. (2012), tannins generally have the property of changing the permeability of membranes and causing cell death.Thus, it may be suggested that the results observed here took place on the basis of these chemical compounds, a condition intensiied with increasing ET.However, further studies should be carried out to deine the action of this plant part and its chemical compounds at the cell level.
Few studies have been conducted to evaluate the cytotoxicity in some species of the genus Hymenaea.Among them, Pettit et al. (2003) stands out, they noticed that the lavonoid palstatina combined with diterpenes extracted from the leaves of Hymenaea palustre has the potential to strongly inhibit cell division of strains from human stomach cancer cells.Likewise, Closa et al. (1997) reported that the lavonoid astilbin and diterpenes extracted from the leaves of Hymenaea martiana signiicantly inhibited cell division in liver cells of rats treated with a clastogenic drug (ClC 4 ), showing a hepatoprotective potential.Abdel-Kader et al. (2002) observed that diterpenes observed on the cork cambium of Hymenaea courbaril were cytotoxic to human ovarian cancer cells, A2780 strain.Therefore, the results obtained by these studies corroborate those obtained by our research group for H. stigonocarpa, i.e. these plants have the ability to inhibit cell proliferation.
Many currently used antitumor drugs were isolated from herbs, such as paclitaxel, vinca alkaloids, and camptothecin (WALL; WANI 1995), something which makes molecular bioprospection a signiicant issue regarding the search for new therapeutic approaches to cancer.The test with A. cepa, despite preliminary, points out new potential antitumor drugs, as observed in this study, thus, there is a need for conducting further studies to evaluate the toxicity of H. stigonocarpa at the cell level by using other test systems, such as testing in cancer cell lines under different exposure times and different treatment regimens, in order to accurately determine the antiproliferative potential of this legume species.

TABLE 1 :
Total number of cells analyzed and the cell cycle phases of A. cepa roots treated with water (control) and with the hydroalcoholic extract of the bark of H. stigonocarpa (0.5; 1.0, and 1.5 mg/mL) at ET 24 h and 48 h.Analysis of cells per group.
ET -Exposure time; CO -Control; P -Prophase, M -Metaphase, A -Anaphase, T -Telophase, MI -Mitotic Index.Means followed by the same letter are not signiicantly different at the 5% level by chi-square test.